Modulation of STAT5 expression

ABSTRACT

Compounds, compositions and methods are provided for modulating the expression of STAT5. The compositions comprise oligonucleotides, targeted to nucleic acid encoding STAT5. Methods of using these compounds for modulation of STAT5 expression and for diagnosis and treatment of diseases and conditions associated with expression of STAT5 are provided.

FIELD OF THE DISCLOSURE

Compositions and methods for modulating the expression of STAT5 aredescribed herein. In particular, the present application relates toantisense compounds, particularly oligonucleotide compounds, which, inpreferred embodiments, hybridize with nucleic acid molecules encodingSTAT5. Such compounds are shown herein to modulate the expression ofSTAT5.

BACKGROUND INFORMATION

Many important cellular processes are regulated by cytokines, hormonesand growth factors which interact with cell-surface receptors. Signaltransducer and activator of transcription (STAT) proteins play a crucialrole in coordinating the response of cells to cytokine receptorstimulation by acting as cytosolic messengers and nuclear transcriptionfactors. Upon cytokine stimulation, STATs are phosphorylated on aconserved tyrosine residue. This phosphorylation can be catalyzed by theJanus (JAK) family kinases, intrinsic cellular receptor kinases or othercellular tyrosine kinases. Activated, phosphorylated STATs then dimerizeand translocate to the nucleus, where they bind to DNA or act with otherDNA binding proteins in multiprotein complexes. These complexes regulategene transcription in a affects a wide range of biological processes,including cell growth and differentiation, the immune response,antiviral activity, and homeostasis (Grimley et al., Cytokine GrowthFactor Rev., 1999, 10, 131–157; Lin et al., Oncogene, 2000, 19,2496–2504).

The STATs were originally discovered as critical players in interferonsignaling mediated by cytokine receptors lacking intrinsic tyrosinekinase domains and employing the JAK kinases. To date, seven STAT familymembers have been described: STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5Band STAT6 (Bromberg, J. Clin. Invest., 2002, 109, 1139–1142). STAT5A(also known as mammary gland factor, MGF) and STAT5B are two distinctlyencoded proteins. STAT5A was originally identified as theprolactin-stimulated ovine gland mammary gland factor MGF (Wakao et al.,Embo J., 1994, 13, 2182–2191), but was subsequently characterized asmember of the STAT family when it was identified as an interleukin-2induced STAT protein (Hou et al., Immunity, 1995, 2, 321–329). STAT5Bwas identified as an additional member of the STAT family that issimilarly induced by interleukin-2 (Lin et al., J. Biol. Chem., 1996,271, 10738–10744). Human STAT5A and STAT5B are both localized tochromosome 17 in the band 17q11.2 and have a very similar genomicorganization (Ambrosio et al., Gene, 2002, 285, 311–318; Lin et al., J.Biol. Chem., 1996, 271, 10738–10744). Human STAT5A and STAT5B share 91%identity at the amino acid level (Lin et al., J. Biol. Chem., 1996, 271,10738–10744).

STAT5A and STAT5B transcripts are ubiquitously expressed in humantissues, including spleen, stomach, brain, skeletal muscle, liver,kidney, lung, placenta, pancreas, heart and small intestine (Ambrosio etal., Gene, 2002, 285, 311–318). STAT5 is activated in response to avariety of cytokines, hormones and growth factors, including prolactin,various interleukins, erythropoietin and granulocyte macrophage-colonystimulating factor. STATS has been implicated in transducing signalsthat affect cell proliferation, differentiation and apoptosis,particularly in the processes of hematopoiesis and immunoregulation,reproduction and lipid metabolism (Grimley et al., Cytokine GrowthFactor Rev., 1999, 10, 131–157).

While STAT5A and STAT5B share a high degree of sequence homology, eachSTAT5 has distinct biological functions. STAT5A-deficient mice developnormally, but mammary lobuloalveolar outgrowth during pregnancy isreduced, and female mice fail to lactate after parturition due todefects in mammary gland differentiation (Liu et al., Genes Dev., 1997,11, 179–186). These results demonstrate that STAT5A is essential foradult mammary gland development and lactogenesis. Targeted disruption ofthe murine STAT5B gene leads to a striking loss of multiple, sexuallydifferentiated responses associated with the sexually dimorphic patternof pituitary growth hormone secretion. Male STAT5B-deficient miceexhibit body growth rates and male-specific liver gene expression levelsthat are decreased relative to wild-type female levels, suggesting thatSTAT5B is necessary for the physiological effects of male growth hormoneon body growth rate and liver gene expression. Only a modest decrease ingrowth rate is seen in STAT5B-deficient females (Udy et al., Proc. Natl.Acad. Sci. USA, 1997, 94, 7239–7244). The phenotypes of the genedisrupted mice correlate with the patterns of expression, with STAT5Ahighly abundant in mouse mammary tissue during lactation and STAT5Bhighly abundant in muscle tissue of virgin and lactating female mice andin male mice (Liu et al., Proc. Natl. Acad. Sci. USA, 1995, 92,8831–8835).

Disruption of both STAT5A and STAT5B results in the phenotypesassociated with disruption of each individual gene and also reveals thatthe STAT5 proteins have redundant functions in response to growthhormone and prolactin. Mice deficient in both STAT5A and STAT5B aresmaller than their wild-type littermates, and the females are infertile.Peripheral T cells from these mice are unable to proliferate in responseto T cell receptor engagement and interleukin-2, suggesting that STAT5plays a role in T cell regulation (Teglund et al., Cell, 1998, 93,841–850).

Each STAT5 gene gives rise to both long and short isoforms. Thesefunctionally distinct isoforms, which are activated in distinctpopulations of cells, are generated not by RNA processing but bySTATS-cleaving protease activity, also limited to distinct populationsof cells. Interleukin-3 activates fill-length STAT5A and STAT5B inmature myeloid cell lines and the c-terminally truncated forms in moreimmature myeloid cell lines (Azam et al., Immunity, 1997, 6, 691–701).These naturally occurring truncated variants can inhibit full-lengthSTAT5 function in cultured mammalian cells but do not affect cell growthrate (Moriggl et al., Mol. Cell. Biol., 1996, 16, 5691–5700; Wang etal., Mol. Cell. Biol., 1996, 16, 6141–6148). Additionally, analternatively spliced form of human STAT5B exists, which uses analternative promoter and 5′ exon within the STAT5B gene. This STAT5Btranscript is found only in placenta tissue (Ambrosio et al., Gene,2002, 285, 311–318). Alternatively spliced forms of rat STAT5A have beenisolated from rat mammary gland, and are designated STAT5A1 and STAT5A2(Kazansky et al., Mol. Endocrinol., 1995, 9, 1598–1609). A STAT5Bisoform that lacks the COOH-terminal 40 amino acids has been isolatedfrom rat liver and designated STAT5BΔ40C (Ripperger et al., J. Biol.Chem., 1995, 270, 29998–30006).

The STAT proteins are not known to contribute directly to cell cyclecheckpoint regulation or DNA repair. However, they contribute totumorigenesis through their involvement in growth factor signaling,apoptosis and angiogenesis. Additionally, because this transcriptionfactor family participates in the immune response, defective STATactivity can compromise immune surveillance and thus promote cancer cellsurvival. STAT5 is commonly found constitutively activated in severalcancers. To date, the most common mechanism for constitutivephosphorylation and activation of STAT proteins is excessive JAK kinaseactivity (Bromberg, J. Clin. Invest., 2002, 109, 1139–1142).

A role for STAT5 in the process of tumor initiation and progression isdemonstrated by the link between constitutive STAT5 activity andcultured cell transformation. STAT5 activation is sufficient fortransformation of hematopoietic precursor cells (Spiekermann et al.,Exp. Hematol., 2002, 30, 262–271). Both STAT5A and STAT5B areconstitutively phosphorylated and are transcriptionally active in K562leukemia cells (Carlesso et al., J. Exp. Med., 1996, 183, 811–820; deGroot et al., Blood, 1999, 94, 1108–1112; Weber-Nordt et al., Blood,1996, 88, 809–816). Additionally, increased constitutive activation ofSTAT5 was detected in transformed human squamous epithelial cellsderived from squamous cell carcinomas of the head and neck. Targeting ofSTAT5B, but not STAT5A, with antisense oligonucleotides inhibited thegrowth of these squamous epithelial cells (Leong et al., Oncogene, 2002,21, 2846–2853).

Abnormal STATS activity is indeed found associated with many cancers,particularly hematopoietic malignancies. Constitutively activated STAT5is found in cell samples taken from patients with T-cell and B-cellacute lymphoblastic leukemia (ALL), adult T-cell leukemia/lymphoma(ATLL), adult T-cell leukemia (ATL), acute myeloid leukemia (AML),chronic myelocytic leukemia (CML) and acute promyelocytic-like leukemia(APL-L) (Arnould et al., Hum. Mol. Genet., 1999, 8, 1741–1749; Carlessoet al., J. Exp. Med., 1996, 183, 811–820; Chai et al., J Immunol., 1997,159, 4720–4728; Gouilleux-Gruart et al., Blood, 1996, 87, 1692–1697;Spiekermann et al., Clin. Cancer Res., 2003, 9, 2140–2150; Takemoto etal., Proc. Nati. Acad. Sci. USA, 1997, 94, 13897–13902; Weber-Nordt etal., Blood, 1996, 88, 809–816). Collectively, these data demonstrate theinvolvement of activated STAT5 in hematopoietic cancers.

One mechanism by which constitutively activated STAT5 may promote cancercell survival is through the inhibition of apoptosis. Introduction of aconstitutively activated STAT5 protects murine T lymphoma cells againstdexamethasone-induced apoptosis (Demoulin et al., J. Biol. Chem., 1999,274, 25855–25861). Conversely, blocking of tyrosine kinase signalingusing a small molecule inhibitor in cells which express BCR/ABL, aconstitutively active tyrosine kinase, inhibited cell growth and inducedapoptosis (Donato et al., Blood, 2001, 97, 2846–2853; Huang et al.,Oncogene, 2002, 21, 8804–8816). Apoptosis was correlated with theinhibition of STAT5 activation. Viral delivery of a dominantly actingSTAT5 mutant to CML primary cells, a CML cell line or prostate cancercells induces cell death, consistent with a role of STAT5 signaling ingrowth and survival of cancer cells (Ahonen et al., J. Biol. Chem.,2003; Huang et al., Oncogene, 2002, 21, 8804–8816).

Inappropriate activation of STAT proteins may also allow cancer cells tosurvive and proliferate in the absence of cytokines and growth factors.STAT5 activation is often observed in correlation with the presence theBCR/ABL chimeric oncogene that results from a chromosomal translocation.The BCR/ABL fusion is found in both CML and ALL (Coffer et al.,Oncogene, 2000, 19, 2511–2522). STAT5 activation in cells derived fromCML patients is strictly dependent on BCR/ABL kinase activity andstrongly correlates with its ability to confer cytokine independentgrowth in hematopoietic cells (Carlesso et al., J. Exp. Med., 1996, 183,811–820; Shuai et al., Oncogene, 1996, 13, 247–254). Constitutivelyactivated STAT5 is also found in several CML-derived cell linesexpressing BCR/ABL. Furthermore, BCR/ABL is expressed in peripheralblood cells from patients with AML, and constitutively activated STAT5was found in one of these AML patients (Chai et al., J. Immunol., 1997,159, 4720–4728). Both the alpha and beta isoforms of STAT5A and STAT5Bare found expressed in cells from AML patients and are proposed to bedue to alternative mRNA splicing rather than to proteolytic cleavage(Xia et al., Cancer Res., 1998, 58, 3173–3180). Additionally, STAT5 is amajor target of other leukemic fusion proteins with protein tyrosinekinase activity, including the TEL-JAK2 and TEL-ABL fusion proteins,which act to inappropriately activate STAT5 (Spiekermann et al., Exp.Hematol., 2002, 30, 262–271).

A case of acute promyelocytic-like leukemia (APL-L) exhibits astructurally abnormal STAT gene that is the result of a fusion betweenthe retinoic acid receptor alpha (RARA) gene and the STAT5B gene.Whereas STAT5B under normal circumstances is translocated to the nucleusonly upon tyrosine kinase activation, the STAT5B/RARA fusion ismislocalized in the nucleus (Arnould et al., Hum. Mol. Genet., 1999, 8,1741–1749). The fusion protein enhances STAT3 activity, which is acharacteristic shared by other APL fusion proteins (Dong and Tweardy,Blood, 2002, 99, 2637–2646).

Phosphorothioate antisense oligodeoxynucleotides, 24 nucleotides inlength, complementary to the start codon of either STAT5A or STAT5B,were used to inhibit STAT5A and STAT5B expression for the purpose ofinvestigating their role in normal hematopoiesis. Downregulation ofSTAT5A or STAT5B following antisense oligodeoxynucleotide treatment hadno effect on the viability, clonogenecity and apoptosis of cord bloodhematopoietic cells (Baskiewicz-Masiuk et al., Cell Mol. Biol. Lett.,2003, 8, 317–331).

The U.S. Pat. No. 5,534,409 discloses and claims an isolated DNA whichhas at least about 90% sequence identity to a nucleotide sequenceencoding mammary gland growth factor (MGF), also known as STAT5. Alsodisclosed are oligonucleotides useful for hybridization for the purposeof isolating cDNA clones encoding MGF (Groner et al., 1996).

The U.S. Pat. No. 5,618,693 claims and discloses an isolated nucleicacid encoding a human STAT5 (hSTAT5). Generally disclosed are nucleicacids for use as hybridization probes, PCR primers and therapeuticnucleic acids, whereby hStat5 nucleic acids are used to modulate,usually reduce, cellular expression or intracellular concentration oravailability of active hStat5 and whereby these therapeutic nucleicacids are typically antisense nucleic acids (McKnight et al., 1997).

Disclosed and claimed in the U.S. Pat. No. 6,160,092 is a crystal of thecore protein of the STAT protein in dimeric form with an 18-mer duplexDNA that contains a binding site for the STAT dimer, as well as methodsof using the structural information in drug discovery and drugdevelopment. Also disclosed are nucleic acid molecules encoding STAT5A,including RNA and DNA molecules and hybridizable nucleic acid moleculeswith minimum length of 12 nucleotides (Chen et al., 2000).

Disclosed in the U.S. Pat. No. 6,518,021 are nucleic acid moleculesencoding a human STAT5A molecule, whereby a nucleic acid molecule is aDNA or RNA sequence or a PCR primer (Thastrup et al., 2003).

The PCT publication WO 02/46466 discloses and claims a method ofinhibiting cellular proliferation mediated by a BRCA/STAT complex,comprising contacting a BRCA/STAT-containing cell with an effectiveamount of a BRCA/STAT complex modulating compound sufficient to modulatethe amount or activity of a BRCA/STAT complex in said cell, wherein saidBRCA/STAT complex modulating compound is selected from the groupconsisting of small molecule, polypeptide and nucleic acid. Thisapplication discloses that a BRCA/STAT complex modulating compound canbe a nucleic acid, such as a DNA or RNA molecule, including antisensenucleic acids, that specifically binds to a BRCA or STAT nucleic acid(Valgeirsdottir, 2002).

The US Pre-grant publication 20030105057 claims and discloses a methodwherein the amount of phosphorylated RECEPTOR/PTK-STAT pathway in a cellis altered by introducing into the cell nucleic acid molecules thatencode RECEPTOR/PTK-STAT proteins, including STAT5A/B, to effect theincrease or decrease of the expression and/or activation of a RECEPTORor STAT, wherein the STAT can be STAT5A/B. This application furtherdiscloses methods whereby the amount of phosphorylated RECEPTOR/PTK-STATprotein is increased or decreased by introducing into the cell anantisense nucleic acid molecule that encodes a tyrosine kinase and/or aRECEPTOR/PTK-STAT protein (Fu et al., 2003).

SUMMARY

Described herein are antisense compounds, especially nucleic acid andnucleic acid-like oligomers, which are targeted to a nucleic acidencoding STAT5, and which modulate the expression of STAT5.Pharmaceutical and other compositions comprising these compounds arealso provided. Further provided are methods of screening for modulatorsof STAT5 and methods of modulating the expression of STAT5 in cells,tissues or animals comprising contacting said cells, tissues or animalswith one or more of the compounds or compositions described herein.Methods of treating an animal, particularly a human, suspected of havingor being prone to a disease or condition associated with expression ofSTAT5 are also set forth herein. Such methods comprise administering atherapeutically or prophylactically effective amount of one or more ofthe compounds or compositions to the person in need of treatment. Inanother embodiment, the antisense compounds disclosed herein optionallyexclude ISIS 130826.

DETAILED DESCRIPTION

A. Overview

The use of standard cytotoxic chemotherapy in the treatment of cancers,in particular leukemias, has reached a plateau; thus, there exists aneed for novel therapies designed to such cancers. Antisense technologyis an effective means for reducing the expression of specific geneproducts and is therefore useful in a number of therapeutic, diagnostic,and research applications for the modulation of STAT5 expression.

Described herein are compositions and methods for modulating STAT5expression (STAT5A and/or STAT5B), including simultaneous modulation ofboth isoforms, STAT5A and STAT5B. This is accomplished by providingoligonucleotides which specifically hybridize with one or more nucleicacid molecules encoding STAT5A, STAT5B or both STAT5A and STAT5B. Asdescribed in more detail in the examples set forth herein, singleoligonucleotides may be used to target STAT5A, STAT5B or both STAT5A andSTAT5B. The oligonucleotides that target both isoforms bind to regionsthat similar nucleotide sequence in both isoforms. In one embodiment,the term “similar” indicates at least about 70%, 75%, 80%, 85%, 90%or >95% identity between the conserved regions in both isoforms.

The antisense oligonucleotides targeted to STAT5 are useful in treatingvarious disorders arising from overexpression of STAT5 such ashyperproliferative disorders, including hematopoietic cancers such asT-cell and B-cell acute lymphoblastic leukemia (ALL), adult T-cellleukemia/lymphoma (ATLL), adult T-cell leukemia (ATL), acute mycloidleukemia (AML), chronic myelocytic leukemia (CML) and acutepromyelocytic-like leukemia (APL-L), lymphomas and solid tumors of theprostate, breast, lung, stomach, intestine, head, neck, pancreas, liver,ovary and spleen.

As used herein, the terms “target nucleic acid” and “nucleic acidmolecule encoding STAT5” have been used for convenience to encompass DNAencoding STAT5, RNA (including pre-mRNA and mRNA or portions thereof)transcribed from such DNA, and also cDNA derived from such RNA. Thehybridization of a compound with its target nucleic acid is generallyreferred to as “antisense”. Consequently, the preferred mechanismbelieved to be included in the practice of some embodiments is referredto herein as “antisense inhibition.” Such antisense inhibition istypically based upon hydrogen bonding-based hybridization ofoligonucleotide strands or segments such that at least one strand orsegment is cleaved, degraded, or otherwise rendered inoperable. In thisregard, it is presently preferred to target specific nucleic acidmolecules and their functions for such antisense inhibition.

The functions of DNA to be interfered with can include replication andtranscription. Replication and transcription, for example, can be froman endogenous cellular template, a vector, a plasmid construct orotherwise. The functions of RNA to be interfered with can includefunctions such as translocation of the RNA to a site of proteintranslation, translocation of the RNA to sites within the cell which aredistant from the site of RNA synthesis, translation of protein from theRNA, splicing of the RNA to yield one or more RNA species, and catalyticactivity or complex formation involving the RNA which may be engaged inor facilitated by the RNA. One preferred result of such interferencewith target nucleic acid function is modulation of the expression ofSTAT5. As used herein, “modulation” and “modulation of expression” meaneither an increase (stimulation) or a decrease (inhibition) in theamount or levels of a nucleic acid molecule encoding the gene, e.g., DNAor RNA. Inhibition is often the preferred form of modulation ofexpression and mRNA is often a preferred target nucleic acid.

As used herein, “hybridization” means the pairing of complementarystrands of oligomeric compounds. In the embodiments described herein,the preferred mechanism of pairing involves hydrogen bonding, which maybe Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding,between complementary nucleoside or nucleotide bases (nucleobases) ofthe strands of oligomeric compounds. For example, adenine and thymineare complementary nucleobases which pair through the formation ofhydrogen bonds. Hybridization can occur under varying circumstances.

An antisense compound is specifically hybridizable when binding of thecompound to the target nucleic acid interferes with the normal functionof the target nucleic acid to cause a loss of activity, and there is asufficient degree of complementarity to avoid non-specific binding ofthe antisense compound to non-target nucleic acid sequences underconditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and under conditions in which assays are performed in thecase of in vitro assays.

As used herein, the phrase “stringent hybridization conditions” or“stringent conditions” refers to conditions under which a compound willhybridize to its target sequence, but to a minimal number of othersequences. Stringent conditions are sequence-dependent and will bedifferent in different circumstances and as described herein. “Stringentconditions” under which oligomeric compounds hybridize to a targetsequence are determined by the nature and composition of the oligomericcompounds and the assays in which they are being investigated.

“Complementary,” as used herein, refers to the capacity for precisepairing between two nucleobases of an oligomeric compound. For example,if a nucleobase at a certain position of an oligonucleotide (anoligomeric compound), is capable of hydrogen bonding with a nucleobaseat a certain position of a target nucleic acid, said target nucleic acidbeing a DNA, RNA, or oligonucleotide molecule, then the position ofhydrogen bonding between the oligonucleotide and the target nucleic acidis considered to be a complementary position. The oligonucleotide andthe further DNA, RNA, or oligonucleotide molecule are complementary toeach other when a sufficient number of complementary positions in eachmolecule are occupied by nucleobases which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of precise pairing orcomplementarity over a sufficient number of nucleobases such that stableand specific binding occurs between the oligonucleotide and a targetnucleic acid.

It is understood in the art that the sequence of an antisense compoundneed not be 100% complementary to that of its target nucleic acid to bespecifically hybridizable. Moreover, an oligonucleotide may hybridizeover one or more segments such that intervening or adjacent segments arenot involved in the hybridization event (e.g., a loop structure orhairpin structure). It is preferred that the antisense compoundsdescribed herein comprise at least 70%, or at least 75%, or at least80%, or at least 85% sequence complementarity to a target region withinthe target nucleic acid, more preferably that they comprise at least 90%sequence complementarity and even more preferably comprise at least 95%or at least 99% sequence complementarity to the target region within thetarget nucleic acid sequence to which they are targeted. For example, anantisense compound in which 18 of 20 nucleobases of the antisensecompound are complementary to a target region, and would thereforespecifically hybridize, would represent 90 percent complementarity. Inthis example, the remaining noncomplementary nucleobases may beclustered or interspersed with complementary nucleobases and need not becontiguous to each other or to complementary nucleobases. As such, anantisense compound which is 18 nucleobases in length having 4 (four)noncomplementary nucleobases which are flanked by two regions ofcomplete complementarity with the target nucleic acid would have 77.8%overall complementarity with the target nucleic acid and would thus fallwithin the scope of the described embodiments. Percent complementarityof an antisense compound with a region of a target nucleic acid can bedetermined routinely using BLAST programs (basic local alignment searchtools) and PowerBLAST programs known in the art (Altschul et al., J.Mol. Biol., 1990, 215, 403–410; Zhang and Madden, Genome Res., 1997, 7,649–656).

Percent homology, sequence identity or complementarity, can bedetermined by, for example, the Gap program (Wisconsin Sequence AnalysisPackage, Version 8 for Unix, Genetics Computer Group, UniversityResearch Park, Madison Wis.), using default settings, which uses thealgorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482–489). Insome preferred embodiments, homology, sequence identity orcomplementarity, between the oligomeric and target is between about 50%to about 60%. In some embodiments, homology, sequence identity orcomplementarity, is between about 60% to about 70%. In preferredembodiments, homology, sequence identity or complementarity, is betweenabout 70% and about 80%. In more preferred embodiments, homology,sequence identity or complementarity, is between about 80% and about90%. In some preferred embodiments, homology, sequence identity orcomplementarity, is about 90%, about 92%, about 94%, about 95%, about96%, about 97%, about 98%, about 99% or about 100%.

B. Compounds

As used herein, antisense compounds include antisense oligomericcompounds, antisense oligonucleotides, ribozymes, external guidesequence (EGS) oligonucleotides, alternate splicers, primers, probes,and other oligomeric compounds which hybridize to at least a portion ofthe target nucleic acid. As such, these compounds may be introduced inthe form of single-stranded, double-stranded, circular or hairpinoligomeric compounds and may contain structural elements such asinternal or terminal bulges or loops. Once introduced to a system, thecompounds may elicit the action of one or more enzymes or structuralproteins to effect modification of the target nucleic acid.

One non-limiting example of such an enzyme is RNAse H, a cellularendonuclease which cleaves the RNA strand of an RNA:DNA duplex. It isknown in the art that single-stranded antisense compounds which are“DNA-like” elicit RNAse H. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide-mediated inhibition of gene expression. Similar roleshave been postulated for other ribonucleases such as those in the RNaseIII and ribonuclease L family of enzymes.

While the preferred form of antisense compound is a single-strandedantisense oligonucleotide, in many species the introduction ofdouble-stranded structures, such as double-stranded RNA (dsRNA)molecules, has been shown to induce potent and specificantisense-mediated reduction of the function of a gene or its associatedgene products. This phenomenon occurs in both plants and animals and isbelieved to have an evolutionary connection to viral defense andtransposon silencing.

The first evidence that dsRNA could lead to gene silencing in animalscame in 1995 from work in the nematode, Caenorhabditis elegans (Guo andKempheus, Cell, 1995, 81, 611–620). Montgomery et al. have shown thatthe primary interference effects of dsRNA are posttranscriptional(Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 15502–15507).The posttranscriptional antisense mechanism defined in Caenorhabditiselegans resulting from exposure to double-stranded RNA (dsRNA) has sincebeen designated RNA interference (RNAi). This term has been generalizedto mean antisense-mediated gene silencing involving the introduction ofdsRNA leading to the sequence-specific reduction of endogenous targetedmRNA levels (Fire et al., Nature, 1998, 391, 806–811). Recently, it hasbeen shown that it is, in fact, the single-stranded RNA oligomers ofantisense polarity of the dsRNAs which are the potent inducers of RNAi(Tijsterman et al., Science, 2002, 295, 694–697).

The antisense compounds also include modified compounds in which adifferent base is present at one or more of the nucleotide positions inthe compound. For example, if the first nucleotide is an adenosine,modified compounds may be produced which contain thymidine, guanosine orcytidine at this position. This may be done at any of the positions ofthe antisense compound. These compounds are then tested using themethods described herein to determine their ability to inhibitexpression of STAT5 mRNA.

As used herein, the term “oligomeric compound” refers to a polymer oroligomer comprising a plurality of monomeric units. As used herein, theterm “oligonucleotide” refers to an oligomer or polymer of ribonucleicacid (RNA) or deoxyribonucleic acid (DNA) or mimetics, chimeras, analogsand homologs thereof. This term includes oligonucleotides composed ofnaturally occurring nucleobases, sugars and covalent intemucleoside(backbone) linkages as well as oligonucleotides having non-naturallyoccurring portions which function similarly. Such modified orsubstituted oligonucleotides are often preferred over native formsbecause of desirable properties such as, for example, enhanced cellularuptake, enhanced affinity for a target nucleic acid and increasedstability in the presence of nucleases.

While oligonucleotides are a preferred form of antisense compound, otherfamilies of antisense compounds are contemplated as well, including butnot limited to oligonucleotide analogs and mimetics such as thosedescribed herein.

The antisense compounds preferably comprise from about 8 to about 80nucleobases (i.e. from about 8 to about 80 linked nucleosides. Compoundsof 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,or 80 nucleobases in length are also contemplated.

In one preferred embodiment, the antisense compounds are 12 to 50nucleobases in length. One having ordinary skill in the art willappreciate that this embodies compounds of 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleobases inlength.

In another preferred embodiment, the antisense compounds are 15 to 30nucleobases in length. One having ordinary skill in the art willappreciate that this embodies compounds of 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length.

Particularly preferred compounds are oligonucleotides from about 12 toabout 50 nucleobases, even more preferably those comprising from about15 to about 30 nucleobases.

Antisense compounds 8–80 nucleobases in length comprising a stretch ofat least eight (8) consecutive nucleobases selected from within theillustrative antisense compounds are considered to be suitable antisensecompounds as well.

Exemplary preferred antisense compounds include oligonucleotidesequences that comprise at least the 8 consecutive nucleobases from the5′-terminus of one of the illustrative preferred antisense compounds(the remaining nucleobases being a consecutive stretch of the sameoligonucleotide beginning immediately upstream of the 5′-terminus of theantisense compound which is specifically hybridizable to the targetnucleic acid and continuing until the oligonucleotide contains about 8to about 80 nucleobases). Similarly preferred antisense compounds arerepresented by oligonucleotide sequences that comprise at least the 8consecutive nucleobases from the 3′-terminus of one of the illustrativepreferred antisense compounds (the remaining nucleobases being aconsecutive stretch of the same oligonucleotide beginning immediatelydownstream of the 3′-terminus of the antisense compound which isspecifically hybridizable to the target nucleic acid and continuinguntil the oligonucleotide contains about 8 to about 80 nucleobases). Itis also understood that preferred antisense compounds may be representedby oligonucleotide sequences that comprise at least 8 consecutivenucleobases from an internal portion of the sequence of an illustrativepreferred antisense compound, and may extend in either or bothdirections until the oligonucleotide contains about 8 to about 80nucleobases.

One having skill in the art armed with the preferred antisense compoundsillustrated herein will be able, without undue experimentation, toidentify further preferred antisense compounds.

C. Oligonucleotide Targets

“Targeting” an antisense compound to a particular nucleic acid moleculecan be a multistep process. The process usually begins with theidentification of a target nucleic acid whose function is to bemodulated. This target nucleic acid may be, for example, a cellular gene(or mRNA transcribed from the gene) whose expression is associated witha particular disorder or disease state, or a nucleic acid molecule froman infectious agent. As described herein, the target nucleic acidencodes STAT5.

The targeting process usually also includes determination of at leastone target region, segment, or site within the target nucleic acid forthe antisense interaction to occur such that the desired effect, e.g.,modulation of expression, will result. As used herein, the term “region”is defined as a portion of the target nucleic acid having at least oneidentifiable structure, function, or characteristic. Within regions oftarget nucleic acids are segments. “Segments” are defined as smaller orsub-portions of regions within a target nucleic acid. “Sites,” as usedherein, are defined as positions within a target nucleic acid.

Since, as is known in the art, the translation initiation codon istypically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in thecorresponding DNA molecule), the translation initiation codon is alsoreferred to as the “AUG codon,” the “start codon” or the “AUG startcodon”. A minority of genes have a translation initiation codon havingthe RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUGhave been shown to function in vivo. Thus, the terms “translationinitiation codon” and “start codon” can encompass many codon sequences,even though the initiator amino acid in each instance is typicallymethionine (in eukaryotes) or formylmethionine (in prokaryotes). It isalso known in the art that eukaryotic and prokaryotic genes may have twoor more alternative start codons, any one of which may be preferentiallyutilized for translation initiation in a particular cell type or tissue,or under a particular set of conditions. As used herein, “start codon”and “translation initiation codon” refer to the codon or codons that areused in vivo to initiate translation of an mRNA transcribed from a geneencoding STAT5, regardless of the sequence(s) of such codons. It is alsoknown in the art that a translation termination codon (or “stop codon”)of a gene may have one of three sequences, i.e., 5′-UAA, 5′-UAG and5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAG and 5′-TGA,respectively).

The terms “start codon region” and “translation initiation codon region”refer to a portion of such an mRNA or gene that encompasses from about25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or3′) from a translation initiation codon. Similarly, the terms “stopcodon region” and “translation termination codon region” refer to aportion of such an mRNA or gene that encompasses from about 25 to about50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from atranslation termination codon. Consequently, the “start codon region”(or “translation initiation codon region”) and the “stop codon region”(or “translation termination codon region”) are all regions which may betargeted effectively with the antisense compounds described herein.

The open reading frame (ORF) or “coding region,” which is known in theart to refer to the region between the translation initiation codon andthe translation termination codon, is also a region which may betargeted effectively. A preferred region is the intragenic regionencompassing the translation initiation or termination codon of the openreading frame (ORF) of a gene.

Other target regions include the 5′ untranslated region (5′UTR), knownin the art to refer to the portion of an mRNA in the 5′ direction fromthe translation initiation codon, and thus including nucleotides betweenthe 5′ cap site and the translation initiation codon of an mRNA (orcorresponding nucleotides on the gene), and the 3′ untranslated region(3′UTR), known in the art to refer to the portion of an mRNA in the 3′direction from the translation termination codon, and thus includingnucleotides between the translation termination codon and 3′ end of anmRNA (or corresponding nucleotides on the gene). The 5′ cap site of anmRNA comprises an N7-methylated guanosine residue joined to the 5′-mostresidue of the mRNA via a 5′-5′ triphosphate linkage. The 5′ cap regionof an mRNA is considered to include the 5′ cap structure itself as wellas the first 50 nucleotides adjacent to the cap site. It is alsopreferred to target the 5′ cap region.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as “introns,” which are excised froma transcript before it is translated. The remaining (and thereforetranslated) regions are known as “exons” and are spliced together toform a continuous mRNA sequence. Targeting splice sites, i.e.,intron-exon junctions or exon-intron junctions, may also be particularlyuseful in situations where aberrant splicing is implicated in disease,or where an overproduction of a particular splice product is implicatedin disease. Aberrant fusion junctions due to rearrangements or deletionsare also preferred target sites. mRNA transcripts produced via theprocess of splicing of two (or more) mRNAs from different gene sourcesare known as “fusion transcripts”. It is also known that introns can beeffectively targeted using antisense compounds targeted to, for example,DNA or pre-mRNA.

It is also known in the art that alternative RNA transcripts can beproduced from the same genomic region of DNA. These alternativetranscripts are generally known as “variants”. More specifically,“pre-mRNA variants” are transcripts produced from the same genomic DNAthat differ from other transcripts produced from the same genomic DNA ineither their start or stop position and contain both intronic and exonicsequence.

Upon excision of one or more exon or intron regions, or portions thereofduring splicing, pre-mRNA variants produce smaller “mRNA variants”.Consequently, mRNA variants are processed pre-mRNA variants and eachunique pre-mRNA variant must always produce a unique mRNA variant as aresult of splicing. These mRNA variants are also known as “alternativesplice variants”. If no splicing of the pre-mRNA variant occurs then thepre-mRNA variant is identical to the mRNA variant.

It is also known in the art that variants can be produced through theuse of alternative signals to start or stop transcription and thatpre-mRNAs and mRNAs can possess more that one start codon or stop codon.Variants that originate from a pre-mRNA or mRNA that use alternativestart codons are known as “alternative start variants” of that pre-mRNAor mRNA. Those transcripts that use an alternative stop codon are knownas “alternative stop variants” of that pre-mRNA or mRNA. One specifictype of alternative stop variant is the “polyA variant” in which themultiple transcripts produced result from the alternative selection ofone of the “polyA stop signals” by the transcription machinery, therebyproducing transcripts that terminate at unique polyA sites. The types ofvariants described herein are also preferred target nucleic acids.

The locations on the target nucleic acid to which the preferredantisense compounds hybridize are hereinbelow referred to as “preferredtarget segments.” As used herein the term “preferred target segment” isdefined as at least an 8-nucleobase portion of a target region to whichan active antisense compound is targeted. While not wishing to be boundby theory, it is presently believed that these target segments representportions of the target nucleic acid which are accessible forhybridization.

While the specific sequences of certain preferred target segments areset forth herein, one of skill in the art will recognize that theseserve only to illustrate and describe particular embodiments. Additionalpreferred target segments may be identified by one having ordinaryskill.

Target segments 8–80 nucleobases in length comprising a stretch of atleast eight (8) consecutive nucleobases selected from within theillustrative preferred target segments are considered to be suitable fortargeting as well.

Target segments can include DNA or RNA sequences that comprise at leastthe 8 consecutive nucleobases from the 5′-terminus of one of theillustrative preferred target segments (the remaining nucleobases beinga consecutive stretch of the same DNA or RNA beginning immediatelyupstream of the 5′-terminus of the target segment and continuing untilthe DNA or RNA contains about 8 to about 80 nucleobases). Similarlypreferred target segments are represented by DNA or RNA sequences thatcomprise at least the 8 consecutive nucleobases from the 3′-terminus ofone of the illustrative preferred target segments (the remainingnucleobases being a consecutive stretch of the same DNA or RNA beginningimmediately downstream of the 3′-terminus of the target segment andcontinuing until the DNA or RNA contains about 8 to about 80nucleobases). It is also understood that preferred antisense targetsegments may be represented by DNA or RNA sequences that comprise atleast 8 consecutive nucleobases from an internal portion of the sequenceof an illustrative preferred target segment, and may extend in either orboth directions until the oligonucleotide contains about 8 to about 80nucleobases. One having skill in the art armed with the preferred targetsegments illustrated herein will be able, without undue experimentation,to identify further preferred target segments.

Once one or more target regions, segments or sites have been identified,antisense compounds are chosen which are sufficiently complementary tothe target, i.e., hybridize sufficiently well and with sufficientspecificity, to give the desired effect.

The oligomeric antisense compounds may also be targeted to regions ofthe STAT5A target nucleobase sequence (e.g., such as those disclosed inExample 13) comprising nucleobases 1–80, 81–160, 161–240, 241–320,321–400, 401–480, 481–560, 561–640, 641–720, 721–800, 801–880, 881–960,961–1040, 1041–1120, 1121–1200, 1201–1280, 1281–1360, 1361–1440,1441–1520, 1521–1600, 1601–1680, 1681–1760, 1761–1840, 1841–1920,1921–2000, 2001–2080, 2081–2160, 2161–2240, 2241–2320, 2321–2400,2401–2480, 2481–2560, 2561–2640, 2641–2720, 2721–2800, 2801–2880,2881–2960, 2961–3040, 3041–3120, 3121–3200, 3201–3280, 3281–3360,3361–3440, 3441–3520, 3521–3600, 3601–3680, 3681–3760, 3761–3840,3841–3920, 3921–4000, 4001–4080, 4081–4160, 4161–4240, 4241–4298, or anycombination thereof. The oligomeric antisense compounds may further betargeted to regions of the STAT5B target nucleobase sequence (e.g., suchas those disclosed in Example 15) comprising nucleobases 1–80, 81–160,161–240, 241–320, 321–400, 401–480, 481–560, 561–640, 641–720, 721–800,801–880, 881–960, 961–1040, 1041–1120, 1121–1200, 1201–1280, 1281–1360,1361–1440, 1441–1520, 1521–1600, 1601–1680, 1681–1760, 1761–1840,1841–1920, 1921–2000, 2001–2080, 2081–2160, 2161–2240, 2241–2320,2321–2400, 2401–2480, 2481–2560, 2561–2640, 2641–2716, or anycombination thereof.

D. Screening and Target Validation

In a further embodiment, the “preferred target segments” identifiedherein may be employed in a screen for additional compounds thatmodulate the expression of STAT5. “Modulators” are those compounds thatdecrease or increase the expression of a nucleic acid molecule encodingSTAT5 and which comprise at least an 8-nucleobase portion which iscomplementary to a preferred target segment. The screening methodcomprises the steps of contacting a preferred target segment of anucleic acid molecule encoding STAT5 with one or more candidatemodulators, and selecting for one or more candidate modulators whichdecrease or increase the expression of a nucleic acid molecule encodingSTAT5. Once it is shown that the candidate modulator or modulators arecapable of modulating (e.g. either decreasing or increasing) theexpression of a nucleic acid molecule encoding STAT5, the modulator maythen be employed in further investigative studies of the function ofSTAT5, or for use as a research, diagnostic, or therapeutic agent.

The preferred target segments may be also be combined with theirrespective complementary antisense compounds to form stabilizeddouble-stranded (duplexed) oligonucleotides.

Such double stranded oligonucleotide moieties have been shown in the artto modulate target expression and regulate translation as well as RNAprocessing via an antisense mechanism. Moreover, the double-strandedmoieties may be subject to chemical modifications (Fire et al., Nature,1998, 391, 806–811; Timmons and Fire, Nature 1998, 395, 854; Timmons etat., Gene, 2001, 263, 103–112; Tabara et al., Science, 1998, 282,430–431; Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95,15502–15507; Tuschl et al., Genes Dev., 1999, 13, 3191–3197; Elbashir etal., Nature, 2001, 411, 494–498; Elbashir et al., Genes Dev. 2001, 15,188–200). For example, such double-stranded moieties have been shown toinhibit the target by the classical hybridization of antisense strand ofthe duplex to the target, thereby triggering enzymatic degradation ofthe target (Tijsterman et al., Science, 2002, 295, 694–697).

The antisense compounds can also be applied in the areas of drugdiscovery and target validation. The use of the compounds and preferredtarget segments identified herein in drug discovery efforts to elucidaterelationships that exist between STAT5 and a disease state, phenotype,or condition is also contemplated. These methods include detecting ormodulating STAT5 comprising contacting a sample, tissue, cell, ororganism with the compounds described herein, measuring the nucleic acidor protein level of STAT5 and/or a related phenotypic or chemicalendpoint at some time after treatment, and optionally comparing themeasured value to a non-treated sample or sample treated with a furthercompound. These methods can also be performed in parallel or incombination with other experiments to determine the function of unknowngenes for the process of target validation or to determine the validityof a particular gene product as a target for treatment or prevention ofa particular disease, condition, or phenotype.

E. Kits, Research Reagents, Diagnostics, and Therapeutics

The antisense compounds can be utilized for diagnostics, therapeutics,prophylaxis and as research reagents and kits. Furthermore, antisenseoligonucleotides, which are able to inhibit gene expression withexquisite specificity, are often used by those of ordinary skill toelucidate the function of particular genes or to distinguish betweenfunctions of various members of a biological pathway.

For use in kits and diagnostics, the compounds described herein, eitheralone or in combination with other compounds or therapeutics, can beused as tools in differential and/or combinatorial analyses to elucidateexpression patterns of a portion or the entire complement of genesexpressed within cells and tissues.

As one nonlimiting example, expression patterns within cells or tissuestreated with one or more antisense compounds are compared to controlcells or tissues not treated with antisense compounds and the patternsproduced are analyzed for differential levels of gene expression as theypertain, for example, to disease association, signaling pathway,cellular localization, expression level, size, structure or function ofthe genes examined. These analyses can be performed on stimulated orunstimulated cells and in the presence or absence of other compoundswhich affect expression patterns.

Examples of methods of gene expression analysis known in the art includeDNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480,17–24; Celis, et al., FEBS Lett., 2000, 480, 2–16), SAGE (serialanalysis of gene expression) (Madden, et al., Drug Discov. Today, 2000,5, 415–425), READS (restriction enzyme amplification of digested cDNAs)(Prashar and Weissman, Methods Enzymol., 1999, 303, 258–72), TOGA (totalgene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci.U.S.A., 2000, 97, 1976–81), protein arrays and proteomics (Celis, etal., FEBS Lett., 2000, 480, 2–16; Jungblut, et al., Electrophoresis,1999, 20, 2100–10), expressed sequence tag (EST) sequencing (Celis, etal., FEBS Lett., 2000, 480, 2–16; Larsson, et al., J. Biotechnol., 2000,80, 143–57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal.Biochem., 2000, 286, 91–98; Larson, et al., Cytometry, 2000, 41,203–208), subtractive cloning, differential display (DD) (Jurecic andBelmont, Curr. Opin. Microbiol., 2000, 3, 316–21), comparative genomichybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31,286–96), FISH (fluorescent in situ hybridization) techniques (Going andGusterson, Eur. J. Cancer, 1999, 35, 1895–904) and mass spectrometrymethods (To, Comb. Chem. High Throughput Screen, 2000, 3, 235–41).

The antisense compounds described herein are useful for research anddiagnostics, because these compounds hybridize to nucleic acids encodingSTAT5. For example, oligonucleotides that are shown to hybridize withsuch efficiency and under such conditions as disclosed herein as to beeffective STAT5 inhibitors will also be effective primers or probesunder conditions favoring gene amplification or detection, respectively.These primers and probes are useful in methods requiring the specificdetection of nucleic acid molecules encoding STAT5 and in theamplification of said nucleic acid molecules for detection or for use infurther studies of STAT5. Hybridization of the antisenseoligonucleotides, particularly the primers and probes described herein,with a nucleic acid encoding STAT5 can be detected by means known in theart. Such means may include conjugation of an enzyme to theoligonucleotide, radiolabelling of the oligonucleotide or any othersuitable detection means. Kits using such detection means for detectingthe level of STATS in a sample may also be prepared.

The specificity and sensitivity of antisense is also harnessed by thoseof skill in the art for therapeutic uses. Antisense compounds have beenemployed as therapeutic moieties in the treatment of disease states inanimals, including humans. Antisense oligonucleotide drugs, includingribozymes, have been safely and effectively administered to humans andnumerous clinical trials are presently underway. It is thus establishedthat antisense compounds can be useful therapeutic modalities that canbe configured to be useful in treatment regimes for the treatment ofcells, tissues and animals, especially humans.

For therapeutics, an animal, preferably a human, suspected of having adisease or disorder which can be treated by modulating the expression ofSTAT5 is treated by administering antisense compounds described herein.For example, in one non-limiting embodiment, the methods comprise thestep of administering to the animal in need of treatment, atherapeutically effective amount of a STAT5 inhibitor. The STAT5inhibitors effectively inhibit the activity of the STAT5 protein orinhibit the expression of the STAT5 protein. In one embodiment, theactivity or expression of STAT5 in an animal is inhibited by about 10%.Preferably, the activity or expression of STAT5 in an animal isinhibited by about 30%. More preferably, the activity or expression ofSTAT5 in an animal is inhibited by 50% or more. Thus, the oligomericantisense compounds modulate expression of STAT5 mRNA by at least 10%,by at least 20%, by at least 25%, by at least 30%, by at least 40%, byat least 50%, by at least 60%, by at least 70%, by at least 75%, by atleast 80%, by at least 85%, by at least 90%, by at least 95%, by atleast 98%, by at least 99%, or by 100%.

For example, the reduction of the expression of STAT5 may be measured inserum, adipose tissue, liver or any other body fluid, tissue or organ ofthe animal. Preferably, the cells contained within said fluids, tissuesor organs being analyzed contain a nucleic acid molecule encoding STAT5protein and/or the STAT5 protein itself.

The antisense compounds are utilized in pharmaceutical compositions byadding an effective amount of a compound to a suitable pharmaceuticallyacceptable diluent or carrier. The compounds and methods describedherein are also useful prophylactically.

F. Modifications

As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base sometimesreferred to as a “nucleobase” or simply a “base”. The two most commonclasses of such heterocyclic bases are the purines and the pyrimidines.Nucleotides are nucleosides that further include a phosphate groupcovalently linked to the sugar portion of the nucleoside. For thosenucleosides that include a pentofuranosyl sugar, the phosphate group canbe linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar. Informing oligonucleotides, the phosphate groups covalently link adjacentnucleosides to one another to form a linear polymeric compound. In turn,the respective ends of this linear polymeric compound can be furtherjoined to form a circular compound, however, linear compounds aregenerally preferred. In addition, linear compounds may have internalnucleobase complementarity and may therefore fold in a manner as toproduce a fully or partially double-stranded compound. Withinoligonucleotides, the phosphate groups are commonly referred to asforming the internucleoside backbone of the oligonucleotide. The normallinkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.

Modified Internucleoside Linkages (Backbones)

Specific examples of preferred antisense compounds includeoligonucleotides containing modified backbones ornon-natural-internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

Preferred modified oligonucleotide backbones containing a phosphorusatom therein include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriaminoalkylphosphotriesters, methyl and other alkylphosphonates including 3′-alkylene phosphonates, 5′-alkylenephosphonates and chiral phosphonates, phosphinates, phosphoramidatesincluding 3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, selenophosphates and boranophosphateshaving normal 3′–5′ linkages, 2′–5′ linked analogs of these, and thosehaving inverted polarity wherein one or more internucleotide linkages isa 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Preferred oligonucleotideshaving inverted polarity comprise a single 3′ to 3′ linkage at the3′-most internucleotide linkage i.e. a single inverted nucleosideresidue which may be abasic (the nucleobase is missing or has a hydroxylgroup in place thereof). Various salts, mixed salts and free acid formsare also included.

Representative United States patents that teach the preparation of theabove phosphorus-containing linkages include, but are not limited to,U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and5,625,050, certain of which are commonly owned with this application,and each of which is herein incorporated by reference.

Preferred modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

Representative United States patents that teach the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain ofwhich are commonly owned with this application, and each of which isherein incorporated by reference.

Modified Sugar and Internucleoside Linkages-Mimetics

In other preferred antisense compounds, e.g., oligonucleotide mimetics,both the sugar and the internucleoside linkage (i.e. the backbone), ofthe nucleotide units are replaced with novel groups. The nucleobaseunits are maintained for hybridization with an appropriate targetnucleic acid. One such compound, an oligonucleotide mimetic that hasbeen shown to have excellent hybridization properties, is referred to asa peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497–1500.

Some embodiments are oligonucleotides with phosphorothioate backbonesand oligonucleosides with heteroatom backbones, and in particular—CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known as a methylene (methylimino)or MMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and—O—N(CH₃)—CH₂—CH₂— [wherein the native phosphodiester backbone isrepresented as —O—P—O—CH₂—] of the above referenced U.S. Pat. No.5,489,677, and the amide backbones of the above referenced U.S. Pat. No.5,602,240. Also preferred are oligonucleotides having morpholinobackbone structures of the above-referenced U.S. Pat. No. 5,034,506.

Modified Sugars

Modified antisense compounds may also contain one or more substitutedsugar moieties. Preferred are antisense compounds, preferably antisenseoligonucleotides, comprising one of the following at the 2′ position:OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; orO-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may besubstituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl andalkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, where n and m are from 1 to about 10. Otherpreferred oligonucleotides comprise one of the following at the 2′position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl,alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl,Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties. Apreferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, alsoknown as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim.Acta, 1995, 78, 486–504) i.e., an alkoxyalkoxy group. A furtherpreferred modification includes 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in exampleshereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₃)₂, also described in examples hereinbelow.

Other preferred modifications include 2′-methoxy (2′-O—CH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl(2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be inthe arabino (up) position or ribo (down) position. A preferred2′-arabino modification is 2′-F. Similar modifications may also be madeat other positions on the oligonucleotide, particularly the 3′ positionof the sugar on the 3′ terminal nucleotide or in 2′–5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Antisense compounds may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. Representative UnitedStates patents that teach the preparation of such modified sugarstructures include, but are not limited to, U.S. Pat. Nos. 4,981,957;5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633;5,792,747; and 5,700,920, certain of which are commonly owned with theinstant application, and each of which is herein incorporated byreference in its entirety.

A further preferred modification of the sugar includes Locked NucleicAcids (LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety.The linkage is preferably a methylene (—CH₂—)_(n) group bridging the 2′oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs andpreparation thereof are described in WO 98/39352 and WO 99/14226.

Natural and Modified Nucleobases

Antisense compounds may also include nucleobase (often referred to inthe art as heterocyclic base or simply as “base”) modifications orsubstitutions. As used herein, “unmodified” or “natural” nucleobasesinclude the purine bases adenine (A) and guanine (G), and the pyrimidinebases thymine (T), cytosine (C) and uracil (U). Modified nucleobasesinclude other synthetic and natural nucleobases such as 5-methylcytosine(5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine,2-aminoadenine, 6-methyl and other alkyl derivatives of adenine andguanine, 2-propyl and other alkyl derivatives of adenine and guanine,2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil andcytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine and other alkynylderivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine,5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol,8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines,5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituteduracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modifiednucleobases include tricyclic pyrimidines such as phenoxazinecytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, pages858–859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosedby Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289–302, Crooke, S. T. and Lebleu, B.,ed., CRC Press, 1993. Certain of these nucleobases are particularlyuseful for increasing the binding affinity of the compounds describedherein. These include 5-substituted pyrimidines, 6-azapyrimidines andN-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine,5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutionshave been shown to increase nucleic acid duplex stability by 0.6–1.2° C.and are presently preferred base substitutions, even more particularlywhen combined with 2′-O-methoxyethyl sugar modifications.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and5,681,941, certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference, andU.S. Pat. No. 5,750,692, which is commonly owned with the instantapplication and also herein incorporated by reference.

Conjugates

Another modification of the antisense compounds involves chemicallylinking to the antisense compound one or more moieties or conjugateswhich enhance the activity, cellular distribution or cellular uptake ofthe oligonucleotide. These moieties or conjugates can include conjugategroups covalently bound to functional groups such as primary orsecondary hydroxyl groups. Conjugate groups include intercalators,reporter molecules, polyamines, polyamides, polyethylene glycols,polyethers, groups that enhance the pharmacodynamic properties ofoligomers, and groups that enhance the pharmacokinetic properties ofoligomers. Typical conjugate groups include cholesterols, lipids,phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone,acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups thatenhance the pharmacodynamic properties include groups that improveuptake, enhance resistance to degradation, and/or strengthensequence-specific hybridization with the target nucleic acid. Groupsthat enhance the pharmacokinetic properties include groups that improveuptake, distribution, metabolism or excretion of the compounds describedherein. Representative conjugate groups are disclosed in InternationalPatent Application PCT/US92/09196, filed Oct. 23, 1992, and U.S. Pat.No. 6,287,860, the entire disclosure of which are incorporated herein byreference. Conjugate moieties include but are not limited to lipidmoieties such as a cholesterol moiety, cholic acid, a thioether, e.g.,hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g.,dodecandiol or undecyl residues, a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or apolyethylene glycol chain, or adamantane acetic acid, a palmityl moiety,or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.Antisense compounds may also be conjugated to active drug substances,for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen,fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine,2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, abenzothiadiazide, chlorothiazide, a diazepine, indomethicin, abarbiturate, a cephalosporin, a sulfa drug, an antidiabetic, anantibacterial or an antibiotic. Oligonucleotide-drug conjugates andtheir preparation are described in U.S. patent application Ser. No.09/334,130 (filed Jun. 15, 1999) which is incorporated herein byreference in its entirety.

Representative United States patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain ofwhich are commonly owned with the instant application, and each of whichis herein incorporated by reference.

Chimeric Compounds

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide.

Other embodiments also include antisense compounds which are chimericcompounds. “Chimeric” antisense compounds or “chimeras” are antisensecompounds, particularly oligonucleotides, which contain two or morechemically distinct regions, each made up of at least one monomer unit,i.e., a nucleotide in the case of an oligonucleotide compound. Chimericantisense oligonucleotides are thus a form of antisense compound. Theseoligonucleotides typically contain at least one region wherein theoligonucleotide is modified so as to confer upon the oligonucleotideincreased resistance to nuclease degradation, increased cellular uptake,increased stability and/or increased binding affinity for the targetnucleic acid. An additional region of the oligonucleotide may serve as asubstrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Byway of example, RNAse H is a cellular endonuclease which cleaves the RNAstrand of an RNA:DNA duplex. Activation of RNase H, therefore, resultsin cleavage of the RNA target, thereby greatly enhancing the efficiencyof oligonucleotide-mediated inhibition of gene expression. The cleavageof RNA:RNA hybrids can, in like fashion, be accomplished through theactions of endoribonucleases, such as RNAseL which cleaves both cellularand viral RNA. Cleavage of the RNA target can be routinely detected bygel electrophoresis and, if necessary, associated nucleic acidhybridization techniques known in the art.

Chimeric antisense compounds may be formed as composite structures oftwo or more oligonucleotides, modified oligonucleotides,oligonucleosides and/or oligonucleotide mimetics as described above.Such compounds have also been referred to in the art as hybrids orgapmers. Representative United States patents that teach the preparationof such hybrid structures include, but are not limited to, U.S. Pat.Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922,certain of which are commonly owned with the instant application, andeach of which is herein incorporated by reference in its entirety.

G. Formulations

The compounds described herein may also be admixed, encapsulated,conjugated or otherwise associated with other molecules, moleculestructures or mixtures of compounds, as for example, liposomes,receptor-targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption-assisting formulations include,but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

The antisense compounds encompass any pharmaceutically acceptable salts,esters, or salts of such esters, or any other compound which, uponadministration to an animal, including a human, is capable of providing(directly or indirectly) the biologically active metabolite or residuethereof.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the compounds described herein:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto. Foroligonucleotides, preferred examples of pharmaceutically acceptablesalts and their uses are further described in U.S. Pat. No. 6,287,860,which is incorporated herein in its entirety.

Another embodiment is pharmaceutical compositions and formulations whichinclude the antisense compounds described herein. The pharmaceuticalcompositions may be administered in a number of ways depending uponwhether local or systemic treatment is desired and upon the area to betreated. Administration may be topical (including ophthalmic and tomucous membranes including vaginal and rectal delivery), pulmonary,e.g., by inhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal), oralor parenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intrathecal or intraventricular,administration. Oligonucleotides with at least one 2′-O-methoxyethylmodification are believed to be particularly useful for oraladministration. Pharmaceutical compositions and formulations for topicaladministration may include transdermal patches, ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable. Coated condoms,gloves and the like may also be useful.

The pharmaceutical formulations, which may conveniently be presented inunit dosage form, may be prepared according to conventional techniqueswell known in the pharmaceutical industry. Such techniques include thestep of bringing into association the active ingredients with thepharmaceutical carrier(s) or excipient(s). In general, the formulationsare prepared by uniformly and intimately bringing into association theactive ingredients with liquid carriers or finely divided solid carriersor both, and then, if necessary, shaping the product.

The compositions may be formulated into any of many possible dosageforms such as, but not limited to, tablets, capsules, gel capsules,liquid syrups, soft gels, suppositories, and enemas. The compositionsmay also be formulated as suspensions in aqueous, non-aqueous or mixedmedia. Aqueous suspensions may further contain substances which increasethe viscosity of the suspension including, for example, sodiumcarboxymethylcellulose, sorbitol and/or dextran. The suspension may alsocontain stabilizers.

Pharmaceutical compositions include, but are not limited to, solutions,emulsions, foams and liposome-containing formulations. Thepharmaceutical compositions and formulations may comprise one or morepenetration enhancers, carriers, excipients or other active or inactiveingredients.

Emulsions are typically heterogenous systems of one liquid dispersed inanother in the form of droplets usually exceeding 0.1 μm in diameter.Emulsions may contain additional components in addition to the dispersedphases, and the active drug which may be present as a solution in eitherthe aqueous phase, oily phase or itself as a separate phase.Microemulsions are also contemplated. Emulsions and their uses are wellknown in the art and are further described in U.S. Pat. No. 6,287,860,which is incorporated herein in its entirety.

Formulations include liposomal formulations. As used herein, the term“liposome” means a vesicle composed of amphiphilic lipids arranged in aspherical bilayer or bilayers. Liposomes are unilamellar ormultilamellar vesicles which have a membrane formed from a lipophilicmaterial and an aqueous interior that contains the composition to bedelivered. Cationic liposomes are positively charged liposomes which arebelieved to interact with negatively charged DNA molecules to form astable complex. Liposomes that are pH-sensitive or negatively-chargedare believed to entrap DNA rather than complex with it. Both cationicand noncationic liposomes have been used to deliver DNA to cells.

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome comprises oneor more glycolipids or is derivatized with one or more hydrophilicpolymers, such as a polyethylene glycol (PEG) moiety. Liposomes andtheir uses are further described in U.S. Pat. No. 6,287,860, which isincorporated herein in its entirety.

The pharmaceutical formulations and compositions may also includesurfactants. The use of surfactants in drug products, formulations andin emulsions is well known in the art. Surfactants and their uses arefurther described in U.S. Pat. No. 6,287,860, which is incorporatedherein in its entirety.

In one embodiment, various penetration enhancers are employed to effectthe efficient delivery of nucleic acids, particularly oligonucleotides.In addition to aiding the diffusion of non-lipophilic drugs across cellmembranes, penetration enhancers also enhance the permeability oflipophilic drugs. Penetration enhancers may be classified as belongingto one of five broad categories, i.e., surfactants, fatty acids, bilesalts, chelating agents, and non-chelating non-surfactants. Penetrationenhancers and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein in its entirety.

One of skill in the art will recognize that formulations are routinelydesigned according to their intended use, i.e. route of administration.

Preferred formulations for topical administration include those in whichthe oligonucleotides are in admixture with a topical delivery agent suchas lipids, liposomes, fatty acids, fatty acid esters, steroids,chelating agents and surfactants. Preferred lipids and liposomes includeneutral (e.g. dioleoylphosphatidyl DOPE ethanolamine,dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline)negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA).

For topical or other administration, oligonucleotides may beencapsulated within liposomes or may form complexes thereto, inparticular to cationic liposomes. Alternatively, oligonucleotides may becomplexed to lipids, in particular to cationic lipids. Preferred fattyacids and esters, pharmaceutically acceptable salts thereof, and theiruses are further described in U.S. Pat. No. 6,287,860, which isincorporated herein in its entirety. Topical formulations are describedin detail in U.S. patent application Ser. No. 09/315,298 filed on May20, 1999, which is incorporated herein by reference in its entirety.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. Preferred oral formulationsare those in which oligonucleotides are administered in conjunction withone or more penetration enhancers surfactants and chelators. Preferredsurfactants include fatty acids and/or esters or salts thereof, bileacids and/or salts thereof. Preferred bile acids/salts and fatty acidsand their uses are further described in U.S. Pat. No. 6,287,860, whichis incorporated herein in its entirety. Also preferred are combinationsof penetration enhancers, for example, fatty acids/salts in combinationwith bile acids/salts. A particularly preferred combination is thesodium salt of lauric acid, capric acid and UDCA. Further penetrationenhancers include polyoxyethylene-9-lauryl ether,polyoxyethylene-20-cetyl ether. Oligonucleotides may be deliveredorally, in granular form including sprayed dried particles, or complexedto form micro or nanoparticles. Oligonucleotide complexing agents andtheir uses are further described in U.S. Pat. No. 6,287,860, which isincorporated herein in its entirety. Oral formulations foroligonucleotides and their preparation are described in detail in U.S.applications Ser. No. 09/108,673 (filed Jul. 1, 1998), Ser. No.09/315,298 (filed May 20, 1999) and Ser. No. 10/071,822, filed Feb. 8,2002, each of which is incorporated herein by reference in theirentirety.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Certain embodiments provide pharmaceutical compositions containing oneor more oligomeric compounds and one or more other chemotherapeuticagents which function by a non-antisense mechanism. Examples of suchchemotherapeutic agents include but are not limited to cancerchemotherapeutic drugs such as daunorubicin, daunomycin, dactinomycin,doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide,ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan,mitomycin C, actinomycin D, mithramycin, prednisone,hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine,hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine,chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). When used with the antisense compounds described herein, suchchemotherapeutic agents may be used individually (e.g., 5-FU andoligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for aperiod of time followed by MTX and oligonucleotide), or in combinationwith one or more other such chemotherapeutic agents (e.g., 5-FU, MTX andoligonucleotide, or 5-FU, radiotherapy and oligonucleotide).Anti-inflammatory drugs, including but not limited to nonsteroidalanti-inflammatory drugs and corticosteroids, and antiviral drugs,including but not limited to ribivirin, vidarabine, acyclovir andganciclovir, may also be combined in compositions described herein.Combinations of antisense compounds and other non-antisense drugs arealso contemplated. Two or more combined compounds may be used togetheror sequentially.

In another related embodiment, the compositions may contain one or moreantisense compounds, particularly oligonucleotides, targeted to a firstnucleic acid and one or more additional antisense compounds targeted toa second nucleic acid target. Alternatively, compositions may containtwo or more antisense compounds targeted to different regions of thesame nucleic acid target. Numerous examples of antisense compounds areknown in the art. Two or more combined compounds may be used together orsequentially.

H. Dosing

The formulation of therapeutic compositions and their subsequentadministration (dosing) is believed to be within the skill of those inthe art. Dosing is dependent on severity and responsiveness of thedisease state to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of the disease state is achieved. Optimal dosing schedulescan be calculated from measurements of drug accumulation in the body ofthe patient. Persons of ordinary skill can easily determine optimumdosages, dosing methodologies and repetition rates. Optimum dosages mayvary depending on the relative potency of individual oligonucleotides,and can generally be estimated based on EC₅₀s found to be effective inin vitro and in vivo animal models. In general, dosage is from 0.01 μgto 100 g per kg of body weight, and may be given once or more daily,weekly, monthly or yearly, or even once every 2 to 20 years. Persons ofordinary skill in the art can easily estimate repetition rates fordosing based on measured residence times and concentrations of the drugin bodily fluids or tissues. Following successful treatment, it may bedesirable to have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 μg to 100 g per kgof body weight, once or more daily, to once every 20 years.

While preferred embodiments have been discussed herein, the followingexamples are meant to be illustrative and not limiting. Each of thereferences, GenBank accession numbers, and the like recited in thepresent application is incorporated herein by reference in its entirety.

EXAMPLES Example 1

Synthesis of Nucleoside Phosphoramidites

The following compounds, including amidites and their intermediates wereprepared as described in U.S. Pat. No. 6,426,220 and published PCT WO02/36743; 5′-O-Dimethoxytrityl-thymidine intermediate for 5-methyl dCamidite, 5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidine intermediate for5-methyl-dC amidite,5′-O-Dimethoxytrityl-2′-deoxy-N4-benzoyl-5-methylcytidine penultimateintermediate for 5-methyl dC amidite,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(5-methyl dC amidite), 2′-Fluorodeoxyadenosine, 2′-Fluorodeoxyguanosine,2′-Fluorouridine, 2′-Fluorodeoxycytidine, 2′-O-(2-Methoxyethyl) modifiedamidites, 2′-O-(2-methoxyethyl)-5-methyluridine intermediate,5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridine penultimate intermediate,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE T amidite),5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidineintermediate,5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methyl-cytidinepenultimate intermediate,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE 5-Me-C amidite),[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁶-benzoyladenosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE A amdite),[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-isobutyrylguanosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE G amidite), 2′-O-(Aminooxyethyl) nucleoside amidites and2′-O-(dimethylamino-oxyethyl) nucleoside amidites,2′-(Dimethylaminooxyethoxy) nucleoside amidites,5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine,5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine,2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine,5′-O-tert-Butyldiphenylsilyl-2′-O-[N,Ndimethylaminooxyethyl]-5-methyluridine,2′-O-(dimethylaminooxyethyl)-5-methyluridine,5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine,5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite],2′-(Aminooxyethoxy) nucleoside amidites,N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite],2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites,2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine,5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyluridine and5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyluridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite.

Example 2

Oligonucleotide and Oligonucleoside Synthesis

The antisense compounds described herein may be conveniently androutinely made through the well-known technique of solid phasesynthesis. Equipment for such synthesis is sold by several vendorsincluding, for example, Applied Biosystems (Foster City, Calif.). Anyother means for such synthesis known in the art may additionally oralternatively be employed. It is well known to use similar techniques toprepare oligonucleotides such as the phosphorothioates and alkylatedderivatives.

Oligonucleotides: Unsubstituted and substituted phosphodiester (P═O)oligonucleotides are synthesized on an automated DNA synthesizer(Applied Biosystems model 394) using standard phosphoramidite chemistrywith oxidation by iodine.

Phosphorothioates (P═S) are synthesized similar to phosphodiesteroligonucleotides with the following exceptions: thiation was effected byutilizing a 10% w/v solution of 3,H-1,2-benzodithiole-3-one 1,1-dioxidein acetonitrile for the oxidation of the phosphite linkages. Thethiation reaction step time was increased to 180 sec and preceded by thenormal capping step. After cleavage from the CPG column and deblockingin concentrated ammonium hydroxide at 55° C. (12–16 hr), theoligonucleotides were recovered by precipitating with >3 volumes ofethanol from a 1 M NH₄OAc solution. Phosphinate oligonucleotides areprepared as described in U.S. Pat. No. 5,508,270, herein incorporated byreference.

Alkyl phosphonate oligonucleotides are prepared as described in U.S.Pat. No. 4,469,863, herein incorporated by reference.

3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared asdescribed in U.S. Pat. Nos. 5,610,289 or 5,625,050, herein incorporatedby reference.

Phosphoramidite oligonucleotides are prepared as described in U.S. Pat.No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated byreference.

Alkylphosphonothioate oligonucleotides are prepared as described inpublished PCT applications PCT/US94/00902 and PCT/US93/06976 (publishedas WO 94/17093 and WO 94/02499, respectively), herein incorporated byreference.

3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared asdescribed in U.S. Pat. No. 5,476,925, herein incorporated by reference.

Phosphotriester oligonucleotides are prepared as described in U.S. Pat.No. 5,023,243, herein incorporated by reference.

Borano phosphate oligonucleotides are prepared as described in U.S. Pat.Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

Oligonucleosides: Methylenemethylimino linked oligonucleosides, alsoidentified as MMI linked oligonucleosides, methylenedimethylhydrazolinked oligonucleosides, also identified as MDH linked oligonucleosides,and methylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone compounds having, for instance, alternating MMIand P═O or P═S linkages are prepared as described in U.S. Pat. Nos.5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of whichare herein incorporated by reference.

Formacetal and thioformacetal linked oligonucleosides are prepared asdescribed in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporatedby reference.

Ethylene oxide linked oligonucleosides are prepared as described in U.S.Pat. No. 5,223,618, herein incorporated by reference.

Example 3

RNA Synthesis

In general, RNA synthesis chemistry is based on the selectiveincorporation of various protecting groups at strategic intermediaryreactions. Although one of ordinary skill in the art will understand theuse of protecting groups in organic synthesis, a useful class ofprotecting groups includes silyl ethers. In particular bulky silylethers are used to protect the 5′-hydroxyl in combination with anacid-labile orthoester protecting group on the 2′-hydroxyl. This set ofprotecting groups is then used with standard solid-phase synthesistechnology. It is important to lastly remove the acid labile orthoesterprotecting group after all other synthetic steps. Moreover, the earlyuse of the silyl protecting groups during synthesis ensures facileremoval when desired, without undesired deprotection of 2′ hydroxyl.

Following this procedure for the sequential protection of the5′-hydroxyl in combination with protection of the 2′-hydroxyl byprotecting groups that are differentially removed and are differentiallychemically labile, RNA oligonucleotides were synthesized.

RNA oligonucleotides are synthesized in a stepwise fashion. Eachnucleotide is added sequentially (3′- to 5′-direction) to a solidsupport-bound oligonucleotide. The first nucleoside at the 3′-end of thechain is covalently attached to a solid support. The nucleotideprecursor, a ribonucleoside phosphoramidite, and activator are added,coupling the second base onto the 5′-end of the first nucleoside. Thesupport is washed and any unreacted 5′-hydroxyl groups are capped withacetic anhydride to yield 5′-acetyl moieties. The linkage is thenoxidized to the more stable and ultimately desired P(V) linkage. At theend of the nucleotide addition cycle, the 5′-silyl group is cleaved withfluoride. The cycle is repeated for each subsequent nucleotide.

Following synthesis, the methyl protecting groups on the phosphates arecleaved in 30 minutes utilizing 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate (S₂Na₂)in DMF. The deprotection solution is washed from the solid support-boundoligonucleotide using water. The support is then treated with 40%methylamine in water for 10 minutes at 55° C. This releases the RNAoligonucleotides into solution, deprotects the exocyclic amines, andmodifies the 2′-groups. The oligonucleotides can be analyzed by anionexchange HPLC at this stage.

The 2′-orthoester groups are the last protecting groups to be removed.The ethylene glycol monoacetate orthoester protecting group developed byDharmacon Research, Inc. (Lafayette, Colo.), is one example of a usefulorthoester protecting group which, has the following importantproperties. It is stable to the conditions of nucleoside phosphoramiditesynthesis and oligonucleotide synthesis. However, after oligonucleotidesynthesis the oligonucleotide is treated with methylamine which not onlycleaves the oligonucleotide from the solid support but also removes theacetyl groups from the orthoesters. The resulting 2-ethyl-hydroxylsubstituents on the orthoester are less electron withdrawing than theacetylated precursor. As a result, the modified orthoester becomes morelabile to acid-catalyzed hydrolysis. Specifically, the rate of cleavageis approximately 10 times faster after the acetyl groups are removed.Therefore, this orthoester possesses sufficient stability in order to becompatible with oligonucleotide synthesis and yet, when subsequentlymodified, permits deprotection to be carried out under relatively mildaqueous conditions compatible with the final RNA oligonucleotideproduct.

Additionally, methods of RNA synthesis are well known in the art(Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996; Scaringe,S. A., et al., J. Am. Chem. Soc., 1998, 120, 11820–11821; Matteucci, M.D. and Caruthers, M. H. J. Am. Chem. Soc., 1981, 103, 3185–3191;Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett., 1981, 22,1859–1862; Dahl, B. J., et al., Acta Chem. Scand., 1990, 44, 639–641;Reddy, M. P., et al., Tetrahedrom Lett., 1994, 25, 4311–4314; Wincott,F. et al., Nucleic Acids Res., 1995, 23, 2677–2684; Griffin, B. E., etal., Tetrahedron, 1967, 23, 2301–2313; Griffin, B. E., et al.,Tetrahedron, 1967, 23, 2315–2331).

RNA antisense compounds (RNA oligonucleotides) can be synthesized by themethods herein or purchased from Dharmacon Research, Inc (Lafayette,Colo.). Once synthesized, complementary RNA antisense compounds can thenbe annealed by methods known in the art to form double stranded(duplexed) antisense compounds. For example, duplexes can be formed bycombining 30 μl of each of the complementary strands of RNAoligonucleotides (50 μM RNA oligonucleotide solution) and 15 μl of 5×annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, 2 mMmagnesium acetate) followed by heating for 1 minute at 90° C., then 1hour at 37° C. The resulting duplexed antisense compounds can be used inkits, assays, screens, or other methods to investigate the role of atarget nucleic acid, or for diagnostic or therapeutic purposes.

Example 4

Synthesis of Chimeric Compounds

Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides can be of several different types.These include a first type wherein the “gap” segment of linkednucleosides is positioned between 5′ and 3′ “wing” segments of linkednucleosides and a second “open end” type wherein the “gap” segment islocated at either the 3′ or the 5′ terminus of the oligomeric compound.Oligonucleotides of the first type are also known in the art as“gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

[2′-O-Me]--[2′-deoxy]--[2′-O—Me] Chimeric PhosphorothioateOligonucleotides

Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligonucleotide segments are synthesized usingan Applied Biosystems automated DNA synthesizer Model 394, as above.Oligonucleotides are synthesized using the automated synthesizer and2′-deoxy-5-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings.The standard synthesis cycle is modified by incorporating coupling stepswith increased reaction times for the5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite. The fully protectedoligonucleotide is cleaved from the support and deprotected inconcentrated ammonia (NH₄OH) for 12–16 hr at 55° C. The deprotectedoligo is then recovered by an appropriate method (precipitation, columnchromatography, volume reduced in vacuo and analyzedspetrophotometrically for yield and for purity by capillaryelectrophoresis and by mass spectrometry.

[2′-O-(2-Methoxyethyl)]--[2′-deoxy]--[2′-O-(Methoxyethyl)] ChimericPhosphorothioate Oligonucleotides

[2′-O-(2-methoxyethyl)]--[2′-deoxy]--[-2′-O-(methoxyethyl)] chimericphosphorothioate oligonucleotides were prepared as per the procedureabove for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methylamidites.

[2′-O-(2-Methoxyethyl)Phosphodiester]--[2′-deoxyPhosphorothioate]--[2′-O-(2-Methoxyethyl) Phosphodiester] ChimericOligonucleotides

[2′-O-(2-methoxyethyl phosphodiester]--[2′-deoxyphosphorothioate]--[2′-O-(methoxyethyl) phosphodiester] chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidationwith iodine to generate the phosphodiester internucleotide linkageswithin the wing portions of the chimeric structures and sulfurizationutilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) togenerate the phosphorothioate internucleotide linkages for the centergap.

Other chimeric oligonucleotides, chimeric oligonucleosides and mixedchimeric oligonucleotides/oligonucleosides are synthesized according toU.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 5

Design and Screening of Duplexed Antisense Compounds Targeting STAT5

A series of nucleic acid duplexes comprising the antisense compoundsdescribed herein and their complements can be designed to target STAT5.The nucleobase sequence of the antisense strand of the duplex comprisesat least an 8-nucleobase portion of an oligonucleotide in Table 1. Theends of the strands may be modified by the addition of one or morenatural or modified nucleobases to form an overhang. The sense strand ofthe dsRNA is then designed and synthesized as the complement of theantisense strand and may also contain modifications or additions toeither terminus. For example, in one embodiment, both strands of thedsRNA duplex would be complementary over the central nucleobases, eachhaving overhangs at one or both termini.

For example, a duplex comprising an antisense strand having the sequenceCGAGAGGCGGACGGGACCG (SEQ ID NO: 214) and having a two-nucleobaseoverhang of deoxythymidine(dT) would have the following structure:

In another embodiment, a duplex comprising an antisense strand havingthe same sequence CGAGAGGCGGACGGGACCG (SEQ ID NO: 214) maybe preparedwith blunt ends (no single stranded overhang) as shown:

RNA strands of the duplex can be synthesized by methods disclosed hereinor purchased from Dharmacon Research Inc., (Lafayette, Colo.). Oncesynthesized, the complementary strands are annealed. The single strandsare aliquotted and diluted to a concentration of 50 μM. Once diluted, 30μL of each strand is combined with 15 uL of a 5× solution of annealingbuffer. The final concentration of said buffer is 100 mM potassiumacetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate. The finalvolume is 75 μL. This solution is incubated for 1 minute at 90° C. andthen centrifuged for 15 seconds. The tube is allowed to sit for 1 hourat 37° C. at which time the dsRNA duplexes are used in experimentation.The final concentration of the dsRNA duplex is 20 μM. This solution canbe stored frozen (−20° C.) and freeze-thawed up to 5 times.

Once prepared, the duplexed antisense compounds are evaluated for theirability to modulate STAT5 expression.

When cells reached 80% confluency, they are treated with duplexedantisense compounds. For cells grown in 96-well plates, wells are washedonce with 200 μL OPTI-MEM-1 reduced-serum medium (Gibco BRL) and thentreated with 130 μL of OPTI-MEM-1 containing 12 μg/mL LIPOFECTIN (GibcoBRL) and the desired duplex antisense compound at a final concentrationof 200 nM. After 5 hours of treatment, the medium is replaced with freshmedium. Cells are harvested 16 hours after treatment, at which time RNAis isolated and target reduction measured by RT-PCR.

Example 6

Oligonucleotide Isolation

After cleavage from the controlled pore glass solid support anddeblocking in concentrated ammonium hydroxide at 55° C. for 12–16 hours,the oligonucleotides or oligonucleosides are recovered by precipitationout of 1 M NH₄OAc with >3 volumes of ethanol. Synthesizedoligonucleotides were analyzed by electrospray mass spectroscopy(molecular weight determination) and by capillary gel electrophoresisand judged to be at least 70% full length material. The relative amountsof phosphorothioate and phosphodiester linkages obtained in thesynthesis was determined by the ratio of correct molecular weightrelative to the −16 amu product (+/−32+/−48). For some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 1991, 266, 18162–18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Example 7

Oligonucleotide Synthesis—96 Well Plate Format

Oligonucleotides were synthesized via solid phase P(III) phosphoramiditechemistry on an automated synthesizer capable of assembling 96 sequencessimultaneously in a 96-well format. Phosphodiester internucleotidelinkages were afforded by oxidation with aqueous iodine.Phosphorothioate internucleotide linkages were generated bysulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyl-diiso-propyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per standard or patented methods. They are utilized as base protectedbeta-cyanoethyldiisopropyl phosphoramidites.

Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55–60° C.) for 12–16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 8

Oligonucleotide Analysis—96-Well Plate Format

The concentration of oligonucleotide in each well was assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products was evaluated by capillaryelectrophoresis (CE) in either the 96-well format (Beckman P/ACE™ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition wasconfirmed by mass analysis of the compounds utilizing electrospray-massspectroscopy. All assay test plates were diluted from the master plateusing single and multi-channel robotic pipettors. Plates were judged tobe acceptable if at least 85% of the compounds on the plate were atleast 85% full length.

Example 9

Cell Culture and Oligonucleotide Treatment

The effect of antisense compounds on target nucleic acid expression canbe tested in any of a variety of cell types provided that the targetnucleic acid is present at measurable levels. This can be routinelydetermined using, for example, PCR or Northern blot analysis. Thefollowing cell types are provided for illustrative purposes, but othercell types can be routinely used, provided that the target is expressedin the cell type chosen. This can be readily determined by methodsroutine in the art, for example Northern blot analysis, ribonucleaseprotection assays, or RT-PCR.

T-24 Cells:

The human transitional cell bladder carcinoma cell line T-24 wasobtained from the American Type Culture Collection (ATCC) (Manassas,Va.). T-24 cells were routinely cultured in complete McCoy's 5A basalmedia (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10%fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin100 units per mL, and streptomycin 100 micrograms per mL (InvitrogenCorporation, Carlsbad, Calif.). Cells were routinely passaged bytrypsinization and dilution when they reached 90% confluence. Cells wereseeded into 96-well plates (Falcon-Primaria #353872) at a density of7000 cells/well for use in RT-PCR analysis.

For Northern blotting or other analysis, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

A549 Cells:

The human lung carcinoma cell line A549 was obtained from the AmericanType Culture Collection (ATCC) (Manassas, Va.). A549 cells wereroutinely cultured in DMEM basal media (Invitrogen Corporation,Carlsbad, Calif.) supplemented with 10% fetal calf serum (InvitrogenCorporation, Carlsbad, Calif.), penicillin 100 units per mL, andstreptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad,Calif.). Cells were routinely passaged by trypsinization and dilutionwhen they reached 90% confluence.

NHDF Cells:

Human neonatal dermal fibroblast (NHDF) were obtained from the CloneticsCorporation (Walkersville, Md.). NHDFs were routinely maintained inFibroblast Growth Medium (Clonetics Corporation, Walkersville, Md.)supplemented as recommended by the supplier. Cells were maintained forup to 10 passages as recommended by the supplier.

HEK Cells:

Human embryonic keratinocytes (HEK) were obtained from the CloneticsCorporation (Walkersville, Md.). HEKs were routinely maintained inKeratinocyte Growth Medium (Clonetics Corporation, Walkersville, Md.)formulated as recommended by the supplier. Cells were routinelymaintained for up to 10 passages as recommended by the supplier.

Treatment With Antisense Compounds:

When cells reached 65–75% confluency, they were treated witholigonucleotide. For cells grown in 96-well plates, wells were washedonce with 100 μL OPTI-MEM™-1 reduced-serum medium (InvitrogenCorporation, Carlsbad, Calif.) and then treated with 130 μL ofOPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™ (Invitrogen Corporation,Carlsbad, Calif.) and the desired concentration of oligonucleotide.Cells are treated and data are obtained in triplicate. After 4–7 hoursof treatment at 37° C., the medium was replaced with fresh medium. Cellswere harvested 16–24 hours after oligonucleotide treatment.

The concentration of oligonucleotide used varies from cell line to cellline. To determine the optimal oligonucleotide concentration for aparticular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations. For human cells thepositive control oligonucleotide is selected from either ISIS 13920(TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1) which is targeted to human H-ras,or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 2) which is targeted tohuman Jun-N-terminal kinase-2 (JNK2). Both controls are2′-O-methoxyethyl gapmers (2′-O-methoxyethyls shown in bold) with aphosphorothioate backbone. For mouse or rat cells the positive controloligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 3, a2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with aphosphorothioate backbone which is targeted to both mouse and rat c-raf.The concentration of positive control oligonucleotide that results in80% inhibition of c-H-ras (for ISIS 13920), JNK2 (for ISIS 18078) orc-raf (for ISIS 15770) mRNA is then utilized as the screeningconcentration for new oligonucleotides in subsequent experiments forthat cell line. If 80% inhibition is not achieved, the lowestconcentration of positive control oligonucleotide that results in 60%inhibition of c-H-ras, JNK2 or c-raf mRNA is then utilized as theoligonucleotide screening concentration in subsequent experiments forthat cell line. If 60% inhibition is not achieved, that particular cellline is deemed as unsuitable for oligonucleotide transfectionexperiments. The concentrations of antisense oligonucleotides usedherein are from 50 nM to 300 nM.

Example 10

Analysis of Oligonucleotide Inhibition of STAT5 Expression

Antisense modulation of STAT5 expression can be assayed in a variety ofways known in the art. For example, STAT5 mRNA levels can be quantitatedby, e.g., Northern blot analysis, competitive polymerase chain reaction(PCR), or real-time PCR (RT-PCR). Real-time quantitative PCR ispresently preferred. RNA analysis can be performed on total cellular RNAor poly(A)+mRNA. The preferred method of RNA analysis is the use oftotal cellular RNA as described in other examples herein. Methods of RNAisolation are well known in the art. Northern blot analysis is alsoroutine in the art. Real-time quantitative (PCR) can be convenientlyaccomplished using the commercially available ABI PRISM™ 7600, 7700, or7900 Sequence Detection System, available from PE-Applied Biosystems,Foster City, Calif. and used according to manufacturer's instructions.

Protein levels of STAT5 can be quantitated in a variety of ways wellknown in the art, such as immunoprecipitation, Western blot analysis(immunoblotting), enzyme-linked immunosorbent assay (ELISA) orfluorescence-activated cell sorting (FACS). Antibodies directed to STAT5can be identified and obtained from a variety of sources, such as theMSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), orcan be prepared via conventional monoclonal or polyclonal antibodygeneration methods well known in the art.

Example 11

Design of Phenotypic Assays for the Use of STAT5 Inhibitors

Phenotypic Assays

Once STAT5 inhibitors have been identified by the methods disclosedherein, the compounds are further investigated in one or more phenotypicassays, each having measurable endpoints predictive of efficacy in thetreatment of a particular disease state or condition.

Phenotypic assays, kits and reagents for their use are well known tothose skilled in the art and are herein used to investigate the roleand/or association of STAT5 in health and disease. Representativephenotypic assays, which can be purchased from any one of severalcommercial vendors, include those for determining cell viability,cytotoxicity, proliferation or cell survival (Molecular Probes, Eugene,Oreg.; PerkinElmer, Boston, Mass.), protein-based assays includingenzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences, FranklinLakes, N.J.; Oncogene Research Products, San Diego, Calif.), cellregulation, signal transduction, inflammation, oxidative processes andapoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglycerideaccumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tubeformation assays, cytokine and hormone assays and metabolic assays(Chemicon International Inc., Temecula, Calif.; Amersham Biosciences,Piscataway, N.J.).

In one non-limiting example, cells determined to be appropriate for aparticular phenotypic assay (i.e., MCF-7 cells selected for breastcancer studies; adipocytes for obesity studies) are treated with STAT5inhibitors identified from the in vitro studies as well as controlcompounds at optimal concentrations which are determined by the methodsdescribed above. At the end of the treatment period, treated anduntreated cells are analyzed by one or more methods specific for theassay to determine phenotypic outcomes and endpoints.

Phenotypic endpoints include changes in cell morphology over time ortreatment dose as well as changes in levels of cellular components suchas proteins, lipids, nucleic acids, hormones, saccharides or metals.Measurements of cellular status which include pH, stage of the cellcycle, intake or excretion of biological indicators by the cell, arealso endpoints of interest.

Analysis of the genotype of the cell (measurement of the expression ofone or more of the genes of the cell) after treatment is also used as anindicator of the efficacy or potency of the STAT5 inhibitors. Hallmarkgenes, or those genes suspected to be associated with a specific diseasestate, condition, or phenotype, are measured in both treated anduntreated cells.

Example 12

RNA Isolation

Poly(A)+mRNA Isolation

Poly(A)+mRNA was isolated according to Miura et al., (Clin. Chem., 1996,42, 1758–1764). Other methods for poly(A)+mRNA isolation are routine inthe art. Briefly, for cells grown on 96-well plates, growth medium wasremoved from the cells and each well was washed with 200 μL cold PBS. 60μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5%NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, theplate was gently agitated and then incubated at room temperature forfive minutes. 55 μL of lysate was transferred to Oligo d(T) coated96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60minutes at room temperature, washed 3 times with 200 μL of wash buffer(10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash,the plate was blotted on paper towels to remove excess wash buffer andthen air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH7.6), preheated to 70° C., was added to each well, the plate wasincubated on a 90° C. hot plate for 5 minutes, and the eluate was thentransferred to a fresh 96-well plate.

Cells grown on 100 mm or other standard plates may be treated similarly,using appropriate volumes of all solutions.

Total RNA Isolation

Total RNA was isolated using an RNEASY 96™ kit and buffers purchasedfrom Qiagen Inc. (Valencia, Calif.) following the manufacturer'srecommended procedures. Briefly, for cells grown on 96-well plates,growth medium was removed from the cells and each well was washed with200 μL cold PBS. 150 μL Buffer RLT was added to each well and the platevigorously agitated for 20 seconds. 150 μL of 70% ethanol was then addedto each well and the contents mixed by pipetting three times up anddown. The samples were then transferred to the RNEASY 96™ well plateattached to a QIAVAC™ manifold fitted with a waste collection tray andattached to a vacuum source. Vacuum was applied for 1 minute. 500 μL ofBuffer RW1 was added to each well of the RNEASY 96™ plate and incubatedfor 15 minutes and the vacuum was again applied for 1 minute. Anadditional 500 μL of Buffer RW1 was added to each well of the RNEASY ₉₆™plate and the vacuum was applied for 2 minutes. 1 mL of Buffer RPE wasthen added to each well of the RNEASY 96™ plate and the vacuum appliedfor a period of 90 seconds. The Buffer RPE wash was then repeated andthe vacuum was applied for an additional 3 minutes. The plate was thenremoved from the QIAVAC™ manifold and blotted dry on paper towels. Theplate was then re-attached to the QIAVAC™ manifold fitted with acollection tube rack containing 1.2 mL collection tubes. RNA was theneluted by pipetting 140 μL of RNAse free water into each well,incubating 1 minute, and then applying the vacuum for 3 minutes.

The repetitive pipetting and elution steps may be automated using aQIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially,after lysing of the cells on the culture plate, the plate is transferredto the robot deck where the pipetting, DNase treatment and elution stepsare carried out.

Example 13

Real-time Quantitative PCR Analysis of STAT5 mRNA Levels

Quantitation of STAT5 mRNA levels was accomplished by real-timequantitative PCR using the ABI PRISM™ 7600, 7700, or 7900 SequenceDetection System (PE-Applied Biosystems, Foster City, Calif.) accordingto manufacturer's instructions. This is a closed-tube, non-gel-based,fluorescence detection system which allows high-throughput quantitationof polymerase chain reaction (PCR) products in real-time. As opposed tostandard PCR in which amplification products are quantitated after thePCR is completed, products in real-time quantitative PCR are quantitatedas they accumulate. This is accomplished by including in the PCRreaction an oligonucleotide probe that anneals specifically between theforward and reverse PCR primers, and contains two fluorescent dyes. Areporter dye (e.g., FAM or JOE, obtained from either PE-AppliedBiosystems, Foster City, Calif., Operon Technologies Inc., Alameda,Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) isattached to the 5′ end of the probe and a quencher dye (e.g., TAMRA,obtained from either PE-Applied Biosystems, Foster City, Calif., OperonTechnologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc.,Coralville, Iowa) is attached to the 3′ end of the probe. When the probeand dyes are intact, reporter dye emission is quenched by the proximityof the 3′ quencher dye. During amplification, annealing of the probe tothe target sequence creates a substrate that can be cleaved by the5′-exonuclease activity of Taq polymerase. During the extension phase ofthe PCR amplification cycle, cleavage of the probe by Taq polymerasereleases the reporter dye from the remainder of the probe (and hencefrom the quencher moiety) and a sequence-specific fluorescent signal isgenerated. With each cycle, additional reporter dye molecules arecleaved from their respective probes, and the fluorescence intensity ismonitored at regular intervals by laser optics built into the ABI PRISMSSequence Detection System. In each assay, a series of parallel reactionscontaining serial dilutions of mRNA from untreated control samplesgenerates a standard curve that is used to quantitate the percentinhibition after antisense oligonucleotide treatment of test samples.

Prior to quantitative PCR analysis, primer-probe sets specific to thetarget gene being measured are evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. In multiplexing, boththe target gene and the internal standard gene GAPDH are amplifiedconcurrently in a single sample. In this analysis, mRNA isolated fromuntreated cells is serially diluted. Each dilution is amplified in thepresence of primer-probe sets specific for GAPDH only, target gene only(“single-plexing”), or both (multiplexing). Following PCR amplification,standard curves of GAPDH and target mRNA signal as a function ofdilution are generated from both the single-plexed and multiplexedsamples. If both the slope and correlation coefficient of the GAPDH andtarget signals generated from the multiplexed samples fall within 10% oftheir corresponding values generated from the single-plexed samples, theprimer-probe set specific for that target is deemed multiplexable. Othermethods of PCR are also known in the art.

PCR reagents were obtained from Invitrogen Corporation, (Carlsbad,Calif.). RT-PCR reactions were carried out by adding 20 μL PCR cocktail(2.5×PCR buffer minus MgCl₂, 6.6 MM MgCl₂, 375 μM each of DATP, dCTP,dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nMof probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5 UnitsMuLV reverse transcriptase, and 2.5×ROX dye) to 96-well platescontaining 30 μL total RNA solution (20–200 ng). The RT reaction wascarried out by incubation for 30 minutes at 48° C. Following a 10 minuteincubation at 95° C. to activate the PLATINUM® Taq, 40 cycles of atwo-step PCR protocol were carried out: 95° C. for 15 seconds(denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

Gene target quantities obtained by real time RT-PCR are normalized usingeither the expression level of GAPDH, a gene whose expression isconstant, or by quantifying total RNA using RiboGreen™ (MolecularProbes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real timeRT-PCR, by being run simultaneously with the target, multiplexing, orseparately. Total RNA is quantified using RiboGreen™ RNA quantificationreagent (Molecular Probes, Inc. Eugene, Oreg.). Methods of RNAquantification by RiboGreen™ are taught in Jones, L. J., et al,(Analytical Biochemistry, 1998, 265, 368–374).

In this assay, 170 μL of RiboGreen™ working reagent (RiboGreen™ reagentdiluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a96-well plate containing 30 μL purified, cellular RNA. The plate is readin a CytoFluor 4000 (PE Applied Biosystems) with excitation at 485 nmand emission at 530 nm.

Probes and primers to human STAT5A were designed to hybridize to a humanSTAT5A sequence, using published sequence information (GenBank accessionnumber NM_(—)003152.2, incorporated herein as SEQ ID NO: 4). For humanSTAT5A the PCR primers were:

-   forward primer: AGAGTGCGCCGAGTCTGTCT (SEQ ID NO: 5) reverse primer:    GCATTGAGTGCCTGCAGTGA (SEQ ID NO: 6) and the PCR probe was:    FAM-TGTCATGGTAGAGACCGAGCCTCT-TAMRA (SEQ ID NO: 7) where FAM is the    fluorescent dye and TAMRA is the quencher dye.

For human GAPDH the PCR primers were:

-   forward primer: GAAGGTGAAGGTCGGAGTC(SEQ ID NO: 8) reverse primer:    GAAGATGGTGATGGGATTTC (SEQ ID NO: 9) and the PCR probe was: 5′    JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO: 10) where JOE is the    fluorescent reporter dye and TAMRA is the quencher dye.

Example 14

Northern Blot Analysis of STAT5 mRNA Levels

Eighteen hours after antisense treatment, cell monolayers were washedtwice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc.,Friendswood, Tex.). Total RNA was prepared following manufacturer'srecommended protocols. Twenty micrograms of total RNA was fractionatedby electrophoresis through 1.2% agarose gels containing 1.1%formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNAwas transferred from the gel to HYBOND™-N+ nylon membranes (AmershamPharmacia Biotech, Piscataway, N.J.) by overnight capillary transferusing a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc.,Friendswood, Tex.). RNA transfer was confirmed by UV visualization.Membranes were fixed by UV cross-linking using a STRATALINKER™ UVCrosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probedusing QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.)using manufacturer's recommendations for stringent conditions.

To detect human STAT5A, a human STAT5A specific probe was prepared byPCR using the forward primer AGAGTGCGCCGAGTCTGTCT (SEQ ID NO: 5) and thereverse primer GCATTGAGTGCCTGCAGTGA (SEQ ID NO: 6). To normalize forvariations in loading and transfer efficiency membranes were strippedand probed for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH)RNA (Clontech, Palo Alto, Calif.).

Hybridized membranes were visualized and quantitated using aPHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics,Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreatedcontrols.

Example 15

Antisense Inhibition of Human STAT5A Expression by ChimericPhosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap

A series of antisense compounds was designed to target different regionsof the human STAT5A RNA, using published sequences for human STAT5A(GenBank accession number NM_(—)003152.2, incorporated herein as SEQ IDNO: 4). The compounds are shown in Table 1. “Target site” indicates thefirst (5′-most) nucleotide number on the particular target sequence towhich the compound binds. All compounds in Table 1 are chimericoligonucleotides (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”.The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines. The compounds were analyzed for their effect onSTAT5A mRNA levels by transfection and quantitative real-time PCR asdescribed in other examples herein. Data are averages from twoexperiments in which T-24 cells were treated with 150 nM of theantisense oligonucleotides. The positive control for each datapoint isidentified in the table by sequence ID number. If present, “N.D.”indicates “no data”.

TABLE 1 Inhibition of human STAT5A mRNA levels by chimericphosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gapTARGET SEQ CONTROL SEQ ID TARGET % ID SEQ ID ISIS # REGION NO SITESEQUENCE INHIB NO NO 130817 Coding 4 1173 gcttggatcctcaggctctc 16 12 1130818 Coding 4 1244 gcttctgctggagggccgtc 43 13 1 130820 Coding 4 1361tgatggtctgctgcttccgc 75 14 1 130821 Coding 4 1559 gcatctcctccactgggccg64 15 1 130824 Coding 4 1676 cggtggctgcaaacttggtc 78 16 1 130825 Coding4 2117 gcacggcaaatggcaccctg 44 17 1 130826 Coding 4 2795gggcctggtccatgtacgtg 36 18 1 153814 3′UTR 4 3681 gagcagctcagaaaccctca 7319 1 153820 3′UTR 4 3895 accaaccctccaagtcccgg 77 20 1 315651 Coding 4810 ccctccaggagctgggtggc 57 21 1 315652 Coding 4 820ctgcaccaggccctccagga 80 22 1 315653 Coding 4 828 tgcagctcctgcaccaggcc 8223 1 315654 Coding 4 837 gccttcttctgcagctcctg 75 24 1 315655 Coding 4859 atcttcccccacctggtgct 56 25 1 315656 Coding 4 869gtaaaaacccatcttccccc 51 26 1 315657 Coding 4 874 cttcagtaaaaacccatctt 4227 1 315658 Coding 4 884 ccagcttgatcttcagtaaa 66 28 1 315659 Coding 4891 tagtgccccagcttgatctt 66 29 1 315660 Coding 4 951cggatgcagcggaccagctc 57 30 1 315661 Coding 4 997 attgttggcttctcggacca 6731 1 315662 Coding 4 1083 accagtcgcagctcctcaaa 55 32 1 315663 Coding 41092 tcctgcgtgaccagtcgcag 78 33 1 315664 Coding 4 1100tctctgtgtcctgcgtgacc 87 34 1 315665 Coding 4 1105 ctcattctctgtgtcctgcg79 35 1 315666 Coding 4 1134 tactcctgagtctgctgcag 88 36 1 315667 Coding4 1149 tactggatgatgaagtactc 63 37 1 315668 Coding 4 1164ctcaggctctcctggtactg 58 38 1 315670 Coding 4 1181 caaactgagcttggatcctc71 39 1 315671 Coding 4 1231 ggccgtctcccggctcagac 38 40 1 315672 Coding4 1236 tggagggccgtctcccggct 83 41 1 315674 Coding 4 1256ccagagacacctgcttctgc 68 42 1 315675 Coding 4 1266 aaccaggcctccagagacac69 43 1 315676 Coding 4 1271 gctgcaaccaggcctccaga 74 44 1 315677 Coding4 1281 tgtgcctcacgctgcaacca 80 45 1 315678 Coding 4 1291ctgcagtgtctgtgcctcac 68 46 1 315679 Coding 4 1301 cgcggtactgctgcagtgtc72 47 1 315680 Coding 4 1319 gcttctcggccagctccacg 74 48 1 315681 Coding4 1324 ctggtgcttctcggccagct 80 49 1 315682 Coding 4 1332agggtcttctggtgcttctc 0 50 1 315683 Coding 4 1337 gctgcagggtcttctggtgc 4651 1 315684 Coding 4 1347 ttccgcagcagctgcagggt 43 52 1 315686 Coding 41367 ccaggatgatggtctgctgc 70 53 1 315687 Coding 4 1373cgtcatccaggatgatggtc 56 54 1 315688 Coding 4 1391 gcttccactggatcagctcg66 55 1 315689 Coding 4 1401 tgctgccgccgcttccactg 82 56 1 315690 Coding4 1443 acgtccaggctgccctcggg 72 57 1 315691 Coding 4 1455caggactgtagcacgtccag 80 58 1 315692 Coding 4 1465 cttctcacaccaggactgta57 59 1 315693 Coding 4 1475 tctcggccaacttctcacac 62 60 1 315694 Coding4 1485 tgccagatgatctcggccaa 69 61 1 315695 Coding 4 1495ctgccggttctgccagatga 72 62 1 315696 Coding 4 1505 tgcggatctgctgccggttc67 63 1 315697 Coding 4 1515 tgctcagccctgcggatctg 76 64 1 315698 Coding4 1525 ctggcagaggtgctcagccc 75 65 1 315699 Coding 4 1536atgggcagctgctggcagag 42 66 1 315701 Coding 4 1570 gacctcggccagcatctcct50 67 1 315702 Coding 4 1591 aatgtccgtgatggtggcgt 74 68 1 315703 Coding4 1600 ggctgagataatgtccgtga 70 69 1 315704 Coding 4 1610tggtcaccagggctgagata 63 70 1 315705 Coding 4 1635 ggctgcttctcaatgatgaa45 71 1 315706 Coding 4 1654 ggtcttcaggacctgaggag 73 72 1 315708 Coding4 1741 gatggtggccttcacctggg 43 73 1 315709 Coding 4 1751gctcactgatgatggtggcc 68 74 1 315710 Coding 4 1761 ttggcctgctgctcactgat80 75 1 315711 Coding 4 1770 agcagagacttggcctgctg 77 76 1 315712 Coding4 1848 gtggcttggtggtactccat 83 77 1 315714 Coding 4 2135gccacagcactttgtcaggc 68 78 1 315715 Coding 4 2169 ttgaatttcatgttgagcgc77 79 1 315716 Coding 4 2189 ggttgctctgcacttcggcc 43 80 1 315717 Coding4 2209 gttctccttggtcaggcccc 66 81 1 315718 Coding 4 2229ttctgcgccaggaacacgag 80 82 1 315719 Coding 4 2249 tgctgctgttgttgaacagt74 83 1 315720 Coding 4 2269 actgtagtcctccaggtggc 43 84 1 315721 Coding4 2277 gacaggccactgtagtcctc 35 85 1 315722 Coding 4 2703tatccatcaacagctttagc 77 86 1 315723 Coding 4 2730 accacttgcttgatctgtgg76 87 1 315724 Coding 4 2740 aaactcagggaccacttgct 80 88 1 315726 Coding4 2843 tctgtgggtacatgttatag 79 89 1

As shown in Table 1, SEQ ID NOs 14, 15, 16, 19, 20, 21, 22, 23, 24, 25,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 41, 42, 43, 44, 45, 46,47, 48, 49, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 68, 69,70, 72, 74, 75, 76, 77, 78, 79, 81, 82, 83, 86, 87, 88 and 89demonstrated at least 55% inhibition of human STAT5A expression in thisassay and are therefore preferred. More preferred are SEQ ID NOs 36, 34,77 and 23. The target regions to which these preferred sequences arecomplementary are herein referred to as “preferred target segments” andare therefore preferred for targeting by compounds described herein.These preferred target segments are shown in Table 2. These sequencesare shown to contain thymine (T) but one of skill in the art willappreciate that thymine (T) is generally replaced by uracil (U) in RNAsequences. The sequences represent the reverse complement of thepreferred antisense compounds shown in Table 1. “Target site” indicatesthe first (5′-most) nucleotide number on the particular target nucleicacid to which the oligonucleotide binds. Also shown in Table 2 is thespecies in which each of the preferred target segments was found.

TABLE 2 Sequence and position of preferred target segments identified inSTAT5A. TARGET REV COMP SITE SEQ ID TARGET OF SEQ SEQ ID ID NO SITESEQUENCE ID ACTIVE IN NO 42321 4 1361 gcggaagcagcagaccatca 14 H. sapiens90 42322 4 1559 cggcccagtggaggagatgc 15 H. sapiens 91 42325 4 1676gaccaagtttgcagccaccg 16 H. sapiens 92 69127 4 3681 tgagggtttctgagctgctc19 H. sapiens 93 69133 4 3895 ccgggacttggagggttggt 20 H. sapiens 94231948 4 810 gccacccagctcctggaggg 21 H. sapiens 95 231949 4 820tcctggagggcctggtgcag 22 H. sapiens 96 231950 4 828 ggcctggtgcaggagctgca23 H. sapiens 97 231951 4 837 caggagctgcagaagaaggc 24 H. sapiens 98231952 4 859 agcaccaggtgggggaagat 25 H. sapiens 99 231955 4 884tttactgaagatcaagctgg 28 H. sapiens 100 231956 4 891 aagatcaagctggggcacta29 H. sapiens 101 231957 4 951 gagctggtccgctgcatccg 30 H. sapiens 102231958 4 997 tggtccgagaagccaacaat 31 H. sapiens 103 231959 4 1083tttgaggagctgcgactggt 32 H. sapiens 104 231960 4 1092ctgcgactggtcacgcagga 33 H. sapiens 105 231961 4 1100ggtcacgcaggacacagaga 34 H. sapiens 106 231962 4 1105cgcaggacacagagaatgag 35 H. sapiens 107 231963 4 1134ctgcagcagactcaggagta 36 H. sapiens 108 231964 4 1149gagtacttcatcatccagta 37 H. sapiens 109 231965 4 1164cagtaccaggagagcctgag 38 H. sapiens 110 231967 4 1181gaggatccaagctcagtttg 39 H. sapiens 111 231969 4 1236agccgggagacggccctcca 41 H. sapiens 112 231971 4 1256gcagaagcaggtgtctctgg 42 H. sapiens 113 231972 4 1266gtgtctctggaggcctggtt 43 H. sapiens 114 231973 4 1271tctggaggcctggttgcagc 44 H. sapiens 115 231974 4 1281tggttgcagcgtgaggcaca 45 H. sapiens 116 231975 4 1291gtgaggcacagacactgcag 46 H. sapiens 117 231976 4 1301gacactgcagcagtaccgcg 47 H. sapiens 118 231977 4 1319cgtggagctggccgagaagc 48 H. sapiens 119 231978 4 1324agctggccgagaagcaccag 49 H. sapiens 120 231983 4 1367gcagcagaccatcatcctgg 53 H. sapiens 121 231984 4 1373gaccatcatcctggatgacg 54 H. sapiens 122 23198S 4 1391cgagctgatccagtggaagc 55 H. sapiens 123 231986 4 1401cagtggaagcggcggcagca 56 H. sapiens 124 231987 4 1443cccgagggcagcctggacgt 57 H. sapiens 125 231988 4 1455ctggacgtgctacagtcctg 58 H. sapiens 126 231989 4 1465tacagtcctggtgtgagaag 59 H. sapiens 127 231990 4 1475gtgtgagaagttggccgaga 60 H. sapiens 128 231991 4 1485ttggccgagatcatctggca 61 H. sapiens 129 231992 4 1495tcatctggcagaaccggcag 62 H. sapiens 130 231993 4 1505gaaccggcagcagatccgca 63 H. sapiens 131 231994 4 1515cagatccgcagggctgagca 64 H. sapiens 132 23199S 4 1525gggctgagcacctctgccag 65 H. sapiens 133 231999 4 1591acgccaccatcacggacatt 68 H. sapiens 134 232000 4 1600tcacggacattatctcagcc 69 H. sapiens 135 232001 4 1610tatctcagccctggtgacca 70 H. sapiens 136 232003 4 1654ctcctcaggtcctgaagacc 72 H. sapiens 137 232006 4 1751ggccaccatcatcagtgagc 74 H. sapiens 138 232007 4 1761atcagtgagcagcaggccaa 75 H. sapiens 139 232008 4 1770cagcaggccaagtctctgct 76 H. sapiens 140 232009 4 1848atggagtaccaccaagccac 77 H. sapiens 141 232011 4 2135gcctgacaaagtgctgtggc 78 H. sapiens 142 232012 4 2169gcgctcaacatgaaattcaa 79 H. sapiens 143 232014 4 2209ggggcctgaccaaggagaac 81 H. sapiens 144 232015 4 2229ctcgtgttcctggcgcagaa 82 H. sapiens 145 232016 4 2249actgttcaacaacagcagca 83 H. sapiens 146 232019 4 2703gctaaagctgttgatggata 86 H. sapiens 147 232020 4 2730ccacagatcaaqcaagtggt 87 H. sapiens 148 232021 4 2740agcaagtggtccctgagttt 88 H. sapiens 149 232023 4 2843ctataacatgtacccacaga 89 H. sapiens 150

As these “preferred target segments” have been found by experimentationto be open to, and accessible for, hybridization with the antisensecompounds described herein, one of skill in the art will recognize or beable to ascertain, using no more than routine experimentation, furtherembodiments that encompass other compounds that specifically hybridizeto these preferred target segments and consequently inhibit theexpression of STAT5.

Antisense compounds include antisense oligomeric compounds, antisenseoligonucleotides, ribozymes, external guide sequence (EGS)oligonucleotides, alternate splicers, primers, probes, and other shortoligomeric compounds which hybridize to at least a portion of the targetnucleic acid.

The compounds described in Table 1 also target the human STAT5B isoform.In a further embodiment, the compounds were analyzed for their effect onSTAT5B mRNA levels by quantitative real-time PCR as described in otherexamples herein. Probes and primers to human STAT5B were designed tohybridize to a human STAT5B sequence, using published sequenceinformation (GenBank accession number U48730.2, incorporated herein asSEQ ID NO: 151). For human STAT5B the PCR primers were:

-   forward primer: CTTCATCTTCACCAGAGGAATCACT (SEQ ID NO: 152) reverse    primer: CTTCACATTATGAGTATTGTTTCAAAAGAG (SEQ ID NO: 153) and the PCR    probe was: FAM-TGTGGATGTTTTAATTCCATGAATC-TAMRA SEQ ID NO: 154) where    FAM is the fluorescent dye and TAMRA is the quencher dye. Data are    averages from two experiments in which T-24 cells were treated with    100 nM of the antisense oligonucleotides described herein. The    positive control for each datapoint is identified in the table by    sequence ID number. If present, “N.D.” indicates “no data”. If    present, “−” indicates that no target site for a particular    antisense compound is found in the referenced target sequence.

TABLE 3 Inhibition of human STAT5B mRNA levels by chimericphosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gapTARGET CONTROL SEQ ID TARGET % SEQ SEQ ID ISIS # REGION NO SITE SEQUENCEINHIB ID NO NO 130817 Coding 151 543 gcttggatcctcaggctctc 76 12 1 130818Coding 151 614 gcttctgctggagggccgtc 76 13 1 130820 Coding 151 731tgatggtctgctgcttccgc 66 14 1 130821 Coding 151 929 gcatctcctccactgggccg53 15 1 130824 Coding — — cggtggctgcaaacttggtc 86 16 1 130825 Coding 1511487 gcacggcaaatggcaccctg 84 17 1 130826 Coding 151 2180gggcctggtccatgtacgtg 69 18 1 153814 3′UTR — — gagcagctcagaaaccctca 3 191 153820 3′UTR — — accaaccctccaagtcccgg 8 20 1 315651 Coding 151 180ccctccaggagctgggtggc 65 21 1 315652 Coding 151 190 ctgcaccaggccctccagga89 22 1 315653 Coding 151 198 tgcagctcctgcaccaggcc 89 23 1 315654 Coding151 207 gccttcttctgcagctcctg 87 24 1 315655 Coding 151 229atcttcccccacctggtgct 45 25 1 315656 Coding 151 239 gtaaaaacccatcttccccc49 26 1 315657 Coding 151 244 cttcagtaaaaacccatctt 39 27 1 315658 Coding151 254 ccagcttgatcttcagtaaa 59 28 1 315659 Coding 151 261tagtgccccagcttgatctt 43 29 1 315660 Coding 151 321 cggatgcagcggaccagctc82 30 1 315661 Coding 151 367 attgttggcttctcggacca 63 31 1 315662 Coding151 453 accagtcgcagctcctcaaa 64 32 1 315663 Coding 151 462tcctgcgtgaccagtcgcag 80 33 1 315664 Coding 151 470 tctctgtgtcctgcgtgacc86 34 1 315665 Coding 151 475 ctcattctctgtgtcctgcg 82 35 1 315666 Coding151 504 tactcctgagtctgctgcag 88 36 1 315667 Coding 151 519tactggatgatgaagtactc 67 37 1 315668 Coding 151 534 ctcaggctctcctggtactg50 38 1 315670 Coding 151 551 caaactgagcttggatcctc 65 39 1 315671 Coding151 601 ggccgtctcccggctcagac 75 40 1 315672 Coding 151 606tggagggccgtctcccggct 88 41 1 315674 Coding 151 626 ccagagacacctgcttctgc65 42 1 315675 Coding 151 636 aaccaggcctccagagacac 60 43 1 315676 Coding151 641 gctgcaaccaggcctccaga 78 44 1 315677 Coding 151 651tgtgcctcacgctgcaacca 81 45 1 315678 Coding 151 661 ctgcagtgtctgtgcctcac80 46 1 315679 Coding 151 671 cgcggtactgctgcagtgtc 82 47 1 315680 Coding151 689 gcttctcggccagctccacg 87 48 1 315681 Coding 151 694ctggtgcttctcggccagct 90 49 1 315682 Coding 151 702 agggtcttctggtgcttctc66 50 1 315683 Coding 151 707 gctgcagggtcttctggtgc 66 51 1 315684 Coding151 717 ttccgcagcagctgcagggt 65 52 1 315686 Coding 151 737ccaggatgatggtctgctgc 85 53 1 315687 Coding 151 743 cgtcatccaggatgatggtc78 54 1 315688 Coding 151 761 gcttccactggatcagctcg 68 55 1 315689 Coding151 771 tgctgccgccgcttccactg 86 56 1 315690 Coding 151 813acgtccaggctgccctcggg 80 57 1 315691 Coding 151 825 caggactgtagcacgtccag71 58 1 315692 Coding 151 835 cttctcacaccaggactgta 55 59 1 315693 Coding151 845 tctcggccaacttctcacac 70 60 1 315694 Coding 151 855tgccagatgatctcggccaa 86 61 1 315695 Coding 151 865 ctgccggttctgccagatga83 62 1 315696 Coding 151 875 tgcggatctgctgccggttc 69 63 1 315697 Coding151 885 tgctcagccctgcggatctg 84 64 1 315698 Coding 151 895ctggcagaggtgctcagccc 76 65 1 315699 Coding 151 906 atgggcagctgctggcagag60 66 1 315701 Coding 151 940 gacctcggccagcatctcct 59 67 1 315702 Coding151 961 aatgtccgtgatggtggcgt 80 68 1 315703 Coding 151 970ggctgagataatgtccgtga 71 69 1 315704 Coding 151 980 tggtcaccagggctgagata79 70 1 315705 Coding 151 1005 ggctgcttctcaatgatgaa 60 71 1 315706Coding 151 1024 ggtcttcaggacctgaggag 78 72 1 315708 Coding 151 1111gatggtggccttcacctggg 67 73 1 315709 Coding 151 1121 gctcactgatgatggtggcc82 74 1 315710 Coding 151 1131 ttggcctgctgctcactgat 0 75 1 315711 Coding151 1140 agcagagacttggcctgctg 58 76 1 315712 Coding 151 1218gtggcttggtggtactccat 90 77 1 315714 Coding 151 1505 gccacagcactttgtcaggc84 78 1 315715 Coding 151 1539 ttgaatttcatgttgagcgc 79 79 1 315716Coding 151 1559 ggttgctctgcacttcggcc 75 80 1 315717 Coding 151 1579gttctccttggtcaggcccc 80 81 1 315718 Coding 151 1599 ttctgcgccaggaacacgag85 82 1 315719 Coding 151 1619 tgctgctgttgttgaacagt 72 83 1 315720Coding 151 1639 actgtagtcctccaggtggc 73 84 1 315721 Coding 151 1647gacaggccactgtagtcctc 74 85 1 315722 Coding 151 2088 tatccatcaacagctttagc68 86 1 315723 Coding 151 2115 accacttgcttgatctgtgg 79 87 1 315724Coding 151 2125 aaactcagggaccacttgct 77 88 1 315726 Coding 151 2228tctgtgggtacatgttatag 78 89 1

As shown in Table 3, SEQ ID NOs 12, 13, 14, 16, 17, 18, 21, 22, 23, 24,30, 31, 32, 33, 34, 35, 36, 37, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 61, 62, 63, 64, 65, 66, 68,69, 70, 71, 72, 73, 74, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88and 89, demonstrated at least 60% inhibition of human STAT5B expressionin this assay and are therefore preferred. More preferred are SEQ ID NOs77, 49, 22 and 23. In another embodiment, the antisense compoundsdisclosed herein optionally exclude ISIS 130826. The target regions towhich these preferred sequences are complementary are herein referred toas “preferred target segments” and are therefore preferred for targetingby compounds described herein.

In another embodiment, a single antisense oligonucleotide is used totarget and inhibit expression of nucleic acid encoding both STAT5A andSTAT5B. For example, ISIS 315652 (SEQ ID NO: 22), 315653 (SEQ ID NO:23), and 315715 (SEQ ID NO: 79) each result in similar inhibition ofexpression of both STAT5 isoforms (Tables 1 and 3). The use of anyantisense compound targeted to STATS which inhibits expression of bothSTATS isoforms by at least about 10% is contemplated.

Example 16

Design of Chimeric Phosphorothioate Oligonucleotides Targeting HumanSTAT5 Having 2′-MOE Wings and a Deoxy Gap

In a further embodiment, additional antisense compounds were designed totarget different regions of the human STAT5 RNA, using publishedsequences for human STAT5A and human STAT5B (GenBank accession numberNM_(—)003152.2, incorporated herein as SEQ ID NO: 4; GenBank accessionnumber U48730.2, incorporated herein as SEQ ID NO: 151). The compoundsare shown in Table 4. “Target site” indicates the first (5′-most)nucleotide number on the particular target sequence to which thecompound binds. The target site for each antisense compound with respectto STAT5A or STAT5B is indicated. All compounds in Table 4 are chimericoligonucleotides (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”.The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines.

TABLE 4 Chimeric phosphorothioate oligonucleotides targeting human STAT5having 2′-MOE wings and a deoxy gap STAT5A STAT5B TARGET TARGET SEQ IDTARGET SEQ ID TARGET SEQ ID ISIS # REGION NO SITE NO SITE SEQUENCE NO130819 Coding 4 1316 151 686 tctcggccagctccacgcgg 155 130822 Coding 41568 151 938 cctcggccagcatctcctcc 156 130823 Coding 4 1589 151 959tgtccgtgatggtggcgttg 157 315669 Coding 4 1169 151 539ggatcctcaggctctcctgg 158 315673 Coding 4 1251 151 621gacacctgcttctgctggag 159 315685 Coding 4 1357 151 727ggtctgctgcttccgcagca 160 315700 Coding 4 1558 151 928catctcctccactgggccgg 161 315707 Coding 4 1668 151 1038gcaaacttggtctgggtctt 162 315713 Coding 4 2115 151 1485acggcaaatggcaccctgcc 163 315725 Coding 4 2792 151 2177cctggtccatgtacgtggcg 164

Example 17

Design of Chimeric Phosphorothioate Oligonucleotides Targeting HumanSTAT5A Having 2′-MOE Wings and a Deoxy Gap

In a further embodiment, additional antisense compounds were designed totarget different regions of the human STAT5A RNA, using publishedsequences for human STAT5A (GenBank accession number NM_(—)003152.2,incorporated herein as SEQ ID NO: 4). The compounds are shown in Table5. “Target site” indicates the first (5′-most) nucleotide number on theparticular target sequence to which the compound binds. All compounds inTable 5 are chimeric oligonucleotides (“gapmers”) 20 nucleotides inlength, composed of a central “gap” region consisting of ten2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by five-nucleotide “wings”. The wings are composed of2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone)linkages are phosphorothioate (P═S) throughout the oligonucleotide. Allcytidine residues are 5-methylcytidines.

TABLE 5 Chimeric phosphorothioate oligonucleotides targeting humanSTAT5A having 2′-MOE wings and a deoxy gap TARGET SEQ SEQ ID TARGET IDISIS # Region NO SITE SEQUENCE NO 153802 3′ UTR 4 3057ccgcttcacattgcatattg 165 153803 3′ UTR 4 3060 cgaccgcttcacattgcata 166153804 3′ UTR 4 3148 tgcacaaggacacacacaca 167 153805 3′ UTR 4 3159ggcgtagctcatgcacaagg 168 153806 3′ UTR 4 3189 gccacatcccaggactgcac 169153807 3′ UTR 4 3281 tgcagtgacagaggctcggt 170 153808 3′ UTR 4 3311ggaggaataggtctggctgc 171 153809 3′ UTR 4 3318 gggcccaggaggaataggtc 172153810 3′ UTR 4 3420 ggcaaagcttctcactccgg 173 153811 3′ UTR 4 3616acgcgctctcatagggttca 174 153812 3′ UTR 4 3640 tgctaaggacatggccgggc 175153813 3′ UTR 4 3669 aaccctcactcaaaccggcg 176 153815 3′ UTR 4 3709gccaagcagccaagcaagga 177 153816 3′ UTR 4 3750 aaacgtgggcaacagcatca 178153817 3′ UTR 4 3807 gagaggcaagcaaagaaggc 179 153818 3′ UTR 4 3863cccaccatatcctagaccca 180 153819 3′ UTR 4 3871 cctgtccacccaccatatcc 181153821 3′ UTR 4 3907 ggaggcaagaggaccaaccc 182 153822 3′ UTR 4 3910ccaggaggcaagaggaccaa 183 153823 3′ UTR 4 3985 ccagattccacaggcacgca 184153824 3′ UTR 4 4023 ccagcggagtcaaaccagat 185 153825 3′ UTR 4 4081gcctcaccagaacacagcca 186 153826 3′ UTR 4 4155 tcttccatggtcagctgccc 187153827 3′ UTR 4 4165 gggctctcaatcttccatgg 188

The antisense compounds in Table 5 were tested for their ability toreduce STAT5A protein expression in the human TF-1 erythroleukemia cellline. TF-1 cells were cultured in RPMI 1640 medium supplemented with 10%heat-inactivated fetal bovine serum (Sigma-Aldrich, St.Louis, Mo.), 10mM Hepes, pH 7.2, 50 μM 2-ME, 2 mM L-glutamine, 100 U/ml penicillin, 100μg/ml streptomycin (all supplements from Invitrogen Corporation,Carlsbad, Calif.). ISIS 153814 (SEQ ID NO: 19) and ISIS 153820 (SEQ IDNO: 20) were also tested. 1×10⁷ cells were transfected with 10 μMconcentration of antisense oligonucleotides by electroporation (48 ohms,1200 microFarads, 175 Volts), using a BTX electroporator (San Diego,Calif.). Cells electroporated in the presence of phosphate-bufferedsaline alone served as controls. Cells were harvested 48 hours followingoligonucleotide treatment, and STAT5A protein levels were assessed usingWestern blot analysis.

Western blot analysis (immunoblot analysis) was carried out usingstandard methods. Cells were washed once with PBS, suspended in Laemmlibuffer (100 μL/well), boiled for 5 minutes and loaded on an 8% SDS-PAGEgel. Gels were run for 1.5 hours at 150 V, and transferred tonitrocellulose for western blotting. Primary antibody directed to STAT5Awas used (Upstate Biotechnology, Inc., Charlottesville, Va.) andvisualized using enhanced chemiluminescence. Bands were visualized usinga PHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

SEQ ID NOs 19, 20, 172, 173, 175, 176, 180, 181 and 182 were able toreduce STAT5A protein expression by at least 30%, demonstrating that thetreatment of cultured cells with antisense compounds interferes withSTAT5A protein expression.

Example 18

Antisense Inhibition of Human STAT5 Expression by ChimericPhosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap

In a further embodiment, additional antisense compounds were designed totarget different regions of the human STAT5B RNA, using publishedsequences for human STAT5B (GenBank accession number U48730.2,incorporated herein as SEQ ID NO: 151). “Target site” indicates thefirst (5′-most) nucleotide number on the particular target sequence towhich the compound binds. The target site for both STAT5 isoforms isindicated for each antisense compound. If present, “−” indicates that notarget site for a particular antisense compound is found in thereferenced target sequence. All compounds in Table 6 are chimericoligonucleotides (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”.The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines.

TABLE 6 Chimeric oligonucleotides targeted to human STAT5A and STAT5Bhaving 2′MOE wings and deoxy gaps STAT5B STAT5A TARGET TARGET SEQ SEQ IDTARGET SEQ ID TARGET ID ISIS # REGION NO SITE NO SITE SEQUENCE NO 168517Start codon 151 1 — — cacagccatggtttacccgg 189 168518 Coding 151 87 — —tgccgcacctcaatgggaaa 190 168519 Coding 151 406 — — ggacatggcatcagcaaggc191 168520 Coding 151 616 4 1246 ctgcttctgctggagggccg 192 168521 Coding151 639 4 1269 tgcaaccaggcctccagaga 193 168522 Coding 151 698 4 1328tcttctggtgcttctcggcc 194 168523 Coding 151 843 4 1473tcggccaacttctcacacca 195 168524 Coding 151 927 4 1557atctcctccactgggccggg 196 168525 Coding 151 1174 — — gccactgtaatcattgcggg197 168526 Coding 151 1358 — — ccagctcatttccaccaaca 198 168527 Coding151 1429 — — cgtcgcattgttgtcctggc 199 168528 Coding 151 1499 4 2129gcactttgtcaggcacggca 200 168529 Coding 151 1625 4 2255ggtggctgctgctgttgttg 201 168530 Coding 151 1657 — — ccaggacacagacaggccac202 168531 Coding 151 1661 — — gggaccaggacacagacagg 203 168532 Coding151 1808 — — ggtcatgggcctgttgcttg 204 168533 Coding 151 1871 — —tgccgccaatttctgagtca 205 168534 Coding 151 1983 — — ttcaagtctcccaagcggtc206 168535 Coding 151 1985 — — aattcaagtctcccaagcgg 207 168536 Coding151 2014 — — tggccgatcaggaaacacgt 208 168537 Coding 151 2282 — —ccattgtgtcctccagatcg 209 168538 Coding 151 2331 — — tgactgtccattggccggcc210 168539 Coding 151 2343 — — tgcgggatccactgactgtc 211 168540 Stopcodon 151 2378 — — tgaagatggagaggtcgcgg 212 168541 3′UTR 151 2512 — —gccaccatgcacagaaacac 213

The compounds in Table 6 were analyzed for their effect on STAT5A andSTAT5B mRNA levels in Molt-4 cells. ISIS 153820 (SEQ ID NO: 20), whichtargets only human STAT5A, was used as a control for the inhibition ofSTAT5A. The human T lymphoblast cell line, Molt-4, was cultured inRPMI-1640 containing 10% fetal bovine serum, 1% L-glutamine, 10 mMHepes, and 5×10⁻⁵ M 2-mercaptoethanol (all culture reagents fromInvitrogen Corporation, Carlsbad, Calif.). 1×10⁷ cells were transfectedwith 10 μM concentration of antisense oligonucleotides byelectroporation (48 ohms, 1200 microFarads, 175 Volts), using a BTXelectroporator (San Diego, Calif.). Cells electroporated in the presenceof phosphate-buffered saline alone served as controls. mRNA levels weremonitored 16 hours following transfection. STAT5A and STAT5B expressionlevels were measured by quantitative real-time PCR as described in otherexamples herein. Data were normalized to control samples. If present,“−” indicates that no target site for a particular antisense compound isfound in the referenced target sequence.

TABLE 7 Antisense inhibition of human STAT5B by chimericoligonucleotides having 2′ MOE wings and deoxy gaps STAT5B STAT5A TARGETTARGET SEQ ISIS SEQ ID TARGET % SEQ ID TARGET % ID # Region NO SITEINHIB NO SITE INHIB NO 153820 3′ UTR — — 0 4 3311 45 20 168517 Startcodon 151 1 18 — — 14 189 168518 Coding 151 87 56 — — 0 190 168519Coding 151 406 43 — — 6 191 168520 Coding 151 616 28 4 1246 0 192 168521Coding 151 639 12 4 1269 10 193 168522 Coding 151 698 66 4 1328 23 194168524 Coding 151 927 81 4 1557 17 196 168525 Coding 151 1174 70 — — 4197 168526 Coding 151 1358 57 — — 5 198 168527 Coding 151 1429 75 — — 22199 168528 Coding 151 1499 78 4 2129 59 200 168529 Coding 151 1625 50 42255 0 201 168530 Coding 151 1657 42 — — 39 202 168531 Coding 151 1661 3— — 7 203 168532 Coding 151 1808 66 — — 16 204 168533 Coding 151 1871 83— — 21 205 168534 Coding 151 1983 49 — — 22 206 168535 Coding 151 198539 — — 3 207 168536 Coding 151 2014 69 — — 11 208 168537 Coding 151 228265 — — 4 209 168538 Coding 151 2331 70 — — 50 210 168539 Coding 151 234375 — — 19 211 168540 Stop codon 151 2378 30 — — 20 212 168541 3′ UTR 1512512 73 — — 0 213

These data demonstrate that SEQ ID NOs 190, 191, 196, 197, 198, 201,204, 205, 207, 208, 209, 211 and 213 exhibit greater than 40% inhibitionof STAT5B and less than 20% inhibition of STAT5A.

1. An antisense compound 16, 17, 18, 19, 20,
 21. 22, 23, 24 or 25nucleobases in length targeted to a nucleic acid molecule encodingSTAT5A (SEQ ID NO: 4) or STAT5B (SEQ ID NO: 151), wherein said compoundhas at least 80% identity with SEQ ID NO:
 36. 2. The antisense compoundof claim 1 which is 18, 19,20, 21 or 22 nucleobases in length and has atleast 90% identity with SEQ ID NO:
 36. 3. The antisense compound ofclaim 2 which is 19, 20 or 21 nucleobases in length and has at least 95%identity with SEQ ID NO:
 36. 4. The antisense compound of claim 1 whichis a chimeric antisense compound.
 5. The antisense compound of claim 1comprising at least one modified internucleoside linkage, sugar moiety,or nucleobase.
 6. The antisense compound of claim 1 comprising at leastone 2′-O-methoxyethyl sugar moiety.
 7. The antisense compound of claim 1comprising at least one phosphorothioate internucleoside linkage.
 8. Theantisense compound of claim 1 wherein at least one cytosine is a5-methylcytosine.